Methods and apparatus that increase sequencing-by-binding efficiency

ABSTRACT

A method of nucleic acid detection including (a) contacting a primed template with a polymerase, nucleotide cognate of a first base type and nucleotide cognate of a second base type under ternary complex stabilizing conditions; (b) contacting the primed template nucleic acid with a polymerase, nucleotide cognate of the first base type and nucleotide cognate of a third base type under ternary complex stabilizing conditions; (c) examining products of (a) and (b) for signals produced by a ternary complex that includes the primed template nucleic acid, polymerase and next correct nucleotide, wherein signals for the product of (a) are ambiguous for the first and second base type, and signals for the product of (b) are ambiguous for the first and third base type; (d) disambiguating signals acquired in (c) to identify the next correct nucleotide.

This application claims the benefit of U.S. Provisional Application No.62/489,610, filed Apr. 25, 2017 and U.S. Provisional Application No.62/526,514, filed Jun. 29, 2017, each of which is hereby incorporated byreference in its entirety.

BACKGROUND

The present disclosure relates generally to molecular analysis anddiagnostics, and has specific applicability to nucleic acid sequencing.

The time required to sequence a human genome has dropped precipitouslyin the last decade. The procedure, which used to take several years andmillions of dollars to perform, can now be completed in a few days, fora few thousand dollars. Although the rate of improvement has beenimpressive, and has indeed outpaced the previous bellwether of rapidinnovation, semiconductor fabrication, the currently availablecommercial methods are still unsatisfactory for many clinicalapplications.

A key clinical hope for sequencing has been to provide importantinformation to develop a reliable diagnosis as to whether a patient hasa deadly disease and, moreover, to provide guidance when choosingbetween expensive or life altering treatment options. For example,sequencing can play a key role in confirming a preliminary cancerdiagnosis and helping the patient decide on treatment options such assurgery, chemotherapy or radiation treatment. Although a few days ofdelay for such confirmation is not likely to adversely impact clinicaloutcome, there is a significant adverse toll on the emotional andpsychological state of the patient who endures the delay.

In other situations, clinical outcome is strongly dependent on a rapiddiagnosis. In a handful of cases, sequencing has been used in neonatalintensive care units to identify mystery diseases in newborn infants andlead doctors to otherwise unrecognized treatment options that savedlives. Nevertheless, too many newborns die every year for lack of atimely diagnosis.

Thus, there exist needs for improvements to the accuracy, speed and costof nucleic acid sequencing. The present invention satisfies these needsand provides related advantages as well.

BRIEF SUMMARY

The present disclosure provides a method of nucleic acid detection, thatincludes the steps of (a) forming a mixture under ternary complexstabilizing conditions, wherein the mixture includes a primed templatenucleic acid, a polymerase and nucleotide cognates of first, second andthird base types in the template; (b) examining the mixture to determinewhether a ternary complex formed; and (c) identifying the next correctnucleotide for the primed template nucleic acid molecule, wherein thenext correct nucleotide is identified as a cognate of the first, secondor third base type if ternary complex is detected in step (b), andwherein the next correct nucleotide is imputed to be a nucleotidecognate of a fourth base type based on the absence of a ternary complexin step (b).

Also provided is a method of nucleic acid detection that includes thesteps of (a) sequentially contacting a primed template nucleic acid withat least two separate mixtures under ternary complex stabilizingconditions, wherein the at least two separate mixtures each include apolymerase and a nucleotide, whereby the sequentially contacting resultsin the primed template nucleic acid being contacted, under the ternarycomplex stabilizing conditions, with nucleotide cognates for first,second and third base type base types in the template; (b) examining theat least two separate mixtures to determine whether a ternary complexformed; and (c) identifying the next correct nucleotide for the primedtemplate nucleic acid molecule, wherein the next correct nucleotide isidentified as a cognate of the first, second or third base type ifternary complex is detected in step (b), and wherein the next correctnucleotide is imputed to be a nucleotide cognate of a fourth base typebased on the absence of a ternary complex in step (b).

The present disclosure further provides a method of nucleic aciddetection that includes the steps of (a) contacting a primed templatenucleic acid with a polymerase and a first mixture of nucleotides underternary complex stabilizing conditions, wherein the first mixtureincludes a nucleotide cognate of a first base type and a nucleotidecognate of a second base type; (b) contacting the primed templatenucleic acid with a polymerase and a second mixture of nucleotides underternary complex stabilizing conditions, wherein the second mixtureincludes a nucleotide cognate of the first base type and a nucleotidecognate of a third base type; (c) examining products of steps (a) and(b) for signals produced by a ternary complex that includes the primedtemplate nucleic acid, a polymerase and a next correct nucleotide,wherein signals acquired for the product of step (a) are ambiguous forthe first and second base type, and wherein signals acquired for theproduct of step (b) are ambiguous for the first and third base type; (d)disambiguating signals acquired in step (c) to identify a base type thatbinds the next correct nucleotide. Optionally, to achieve disambiguation(i) the first base type is correlated with presence of signals for theproduct of step (a) and presence of signals for the product of step (b),(ii) the second base type is correlated with presence of signals for theproduct of step (a) and absence of signals for the product of step (b),and (iii) the third base type is correlated with absence of signals forthe product of step (a) and presence of signals for the product of step(b).

Also provided is a method of nucleic acid detection that includes thesteps of (a) contacting a primed template nucleic acid with a firstmixture including a polymerase, a nucleotide cognate of a first basetype in the template and a nucleotide cognate a second base type in thetemplate, wherein the contact occurs in a binding reaction that (i)stabilizes ternary complexes including the primed template nucleic acid,the polymerase and a next correct nucleotide, and (ii) preventsincorporation of the next correct nucleotide into the primer; (b)examining the binding reaction to determine whether a ternary complexformed; (c) subjecting the primed template nucleic acid to a repetitionof steps (a) and (b), wherein the first mixture is replaced with asecond mixture, the second mixture including a polymerase, a nucleotidecognate of the first base type in the template and a nucleotide cognateof a third base type in the template; and (d) identifying the nextcorrect nucleotide for the primed template nucleic acid using theexamination of the binding reaction, or the product thereof, wherein (i)the next correct nucleotide is identified as a cognate of the first basetype if ternary complex is detected in step (b) and detected in therepetition of step (b), (ii) the next correct nucleotide is identifiedas a cognate of the second base type if ternary complex is detected instep (b) and undetected in the repetition of step (b), and (iii) thenext correct nucleotide is identified as a cognate of the third basetype if ternary complex is undetected in step (b) and detected in therepetition of step (b).

In particular embodiments, the steps of a nucleic acid detection methodset forth herein can be repeated to interrogate several differentpositions in a template nucleic acid. Accordingly, this disclosureprovides a method for sequencing a nucleic acid that includes the stepsof (a) forming a mixture under ternary complex stabilizing conditions,wherein the mixture includes a primed template nucleic acid, apolymerase and nucleotide cognates of first, second and third base typesin the template; (b) examining the mixture to determine whether aternary complex formed; (c) identifying the next correct nucleotide forthe primed template nucleic acid molecule, wherein the next correctnucleotide is identified as a cognate of the first, second or third basetype if ternary complex is detected in step (b), and wherein the nextcorrect nucleotide is imputed to be a nucleotide cognate of a fourthbase type based on the absence of a ternary complex in step (b); (d)adding a next correct nucleotide to the primer of the primed templatenucleic acid after step (b), thereby producing an extended primer; and(e) repeating steps (a) through (d) for the primed template nucleic acidthat comprises the extended primer.

In particular embodiments, the steps of a nucleic acid detection methodset forth herein can be repeated to interrogate several differentpositions in a template nucleic acid. Accordingly, this disclosureprovides a method for sequencing a nucleic acid that includes the stepsof (a) forming a mixture under ternary complex stabilizing conditions,wherein the mixture includes a primed template nucleic acid, apolymerase and nucleotide cognates of first, second and third base typesin the template, wherein each nucleotide cognate of first, second andthird base type in the template is capable of forming a ternary complexthat is differentially detectable (i.e., a ternary complex formed with anucleotide cognate of the first, second or third base type, respectivelymay be identified as such and may be identified as different from aternary complex formed with a nucleotide cognate of the second or third,first or third, or first or second base types, respectively); (b)examining the mixture to determine whether a ternary complex formed; (c)identifying the next correct nucleotide for the primed template nucleicacid molecule, wherein the next correct nucleotide is identified as acognate of the first, second or third base type if ternary complex isdetected in step (b), and wherein the next correct nucleotide is imputedto be a nucleotide cognate of a fourth base type based on the absence ofa ternary complex in step (b); (d) adding a next correct nucleotide tothe primer of the primed template nucleic acid after step (b), therebyproducing an extended primer; and (e) repeating steps (a) through (d)for the primed template nucleic acid that comprises the extended primer.

Also provided by this disclosure is a method for sequencing a nucleicacid that includes the steps of (a) sequentially contacting a primedtemplate nucleic acid with at least two separate mixtures under ternarycomplex stabilizing conditions, wherein the at least two separatemixtures each include a polymerase and a nucleotide, whereby thesequentially contacting results in the primed template nucleic acidbeing contacted, under the ternary complex stabilizing conditions, withnucleotide cognates for first, second and third base type base types inthe template; (b) examining the at least two separate mixtures todetermine whether a ternary complex formed; and (c) identifying the nextcorrect nucleotide for the primed template nucleic acid molecule,wherein the next correct nucleotide is identified as a cognate of thefirst, second or third base type if ternary complex is detected in step(b), and wherein the next correct nucleotide is imputed to be anucleotide cognate of a fourth base type based on the absence of aternary complex in step (b); (d) adding a next correct nucleotide to theprimer of the primed template nucleic acid after step (b), therebyproducing an extended primer; and (e) repeating steps (a) through (d)for the primed template nucleic acid that comprises the extended primer.

In further embodiments a method of nucleic acid sequencing can includethe steps of (a) contacting a primed template nucleic acid with apolymerase and a first mixture of nucleotides under ternary complexstabilizing conditions, wherein the first mixture includes a nucleotidecognate of a first base type and a nucleotide cognate of a second basetype; (b) contacting the primed template nucleic acid with a polymeraseand a second mixture of nucleotides under ternary complex stabilizingconditions, wherein the second mixture includes a nucleotide cognate ofthe first base type and a nucleotide cognate of a third base type; (c)examining products of steps (a) and (b) for signals produced by aternary complex that includes the primed template nucleic acid, apolymerase and a next correct nucleotide, wherein signals acquired forthe product of step (a) are ambiguous for the first and second basetype, and wherein signals acquired for the product of step (b) areambiguous for the first and third base type; (d) disambiguating signalsacquired in step (c) to identify a base type that binds the next correctnucleotide; (e) adding a next correct nucleotide to the primer of theprimed template nucleic acid after step (c), thereby producing anextended primer; and (f) repeating steps (a) through (e) for the primedtemplate nucleic acid that comprises the extended primer.

Further still, a method of nucleic acid sequencing can include the stepsof (a) contacting a primed template nucleic acid with a first mixtureincluding a polymerase, a nucleotide cognate of a first base type in thetemplate and a nucleotide cognate a second base type in the template,wherein the contact occurs in a binding reaction that (i) stabilizesternary complexes including the primed template nucleic acid, thepolymerase and a next correct nucleotide, and (ii) preventsincorporation of the next correct nucleotide into the primer; (b)examining the binding reaction to determine whether a ternary complexformed; (c) subjecting the primed template nucleic acid to a repetitionof steps (a) and (b), wherein the first mixture is replaced with asecond mixture, the second mixture including a polymerase, a nucleotidecognate of the first base type in the template and a nucleotide cognateof a third base type in the template; (d) identifying the next correctnucleotide for the primed template nucleic acid using the examination ofthe binding reaction, or the product thereof, wherein (i) the nextcorrect nucleotide is identified as a cognate of the first base type ifternary complex is detected in step (b) and detected in the repetitionof step (b), (ii) the next correct nucleotide is identified as a cognateof the second base type if ternary complex is detected in step (b) andundetected in the repetition of step (b), and (iii) the next correctnucleotide is identified as a cognate of the third base type if ternarycomplex is undetected in step (b) and detected in the repetition of step(b); (e) adding a next correct nucleotide to the primer of the primedtemplate nucleic acid after step (c), thereby producing an extendedprimer; and (f) repeating steps (a) through (e) for the primed templatenucleic acid that comprises the extended primer.

In embodiments, when a ternary complex is detected/undetected in step(b) and detected/undetected in the repetition of step (b), the ternarycomplex is detected/undetected in the first iteration of step (b) anddetected/undetected in the repetition (i.e., second iteration) of step(b).

This disclosure further provides a method of nucleic acid detection thatincludes steps of (a) sequentially contacting a primed template nucleicacid with at least four separate mixtures under ternary complexstabilizing conditions, wherein each of the mixtures includes apolymerase and nucleotide cognates for at least two of four differentbase types in the primed template nucleic acid; (b) examining the atleast four separate mixtures to detect ternary complexes; and (c)identifying the next correct nucleotide for the primed template nucleicacid molecule, wherein the next correct nucleotide is identified as acognate of one of the four different base types if ternary complex isdetected in at least two of the mixtures.

In particular embodiments, a method of nucleic acid detection, caninclude (a) sequentially contacting a primed template nucleic acid withfirst and second mixtures under ternary complex stabilizing conditions,wherein each of the mixtures includes a polymerase and nucleotidecognates for at least two of four different base types in the primedtemplate nucleic acid, wherein the mixtures differ by at least one typeof nucleotide cognate; (b) examining the first and second mixturesseparately to detect ternary complexes; and (c) identifying the nextcorrect nucleotide for the primed template nucleic acid molecule,wherein the next correct nucleotide is identified as a cognate of one ofthe four different base types if ternary complex is detected in the twomixtures.

In particular embodiments, a method of nucleic acid detection, caninclude (a) sequentially contacting a primed template nucleic acid withfirst and second mixtures under ternary complex stabilizing conditions,wherein each of the mixtures includes a polymerase and nucleotidecognates for at least two of four different base types in the primedtemplate nucleic acid, wherein the mixtures differ by at least one typeof nucleotide cognate; (b) examining the first and second mixturesseparately to detect ternary complexes; and (c) identifying the nextcorrect nucleotide for the primed template nucleic acid molecule,wherein the next correct nucleotide is identified as a cognate of one ofthe four different base types if ternary complex is detected in firstmixture but not the second mixture.

In particular embodiments, a method of nucleic acid detection, caninclude (a) sequentially contacting a primed template nucleic acid withfirst and second mixtures under ternary complex stabilizing conditions,wherein each of the mixtures includes a polymerase and nucleotidecognates for at least two of four different base types in the primedtemplate nucleic acid, wherein the mixtures differ by at least one typeof nucleotide cognate; (b) examining the first and second mixturesseparately to detect ternary complexes; and (c) identifying the nextcorrect nucleotide for the primed template nucleic acid molecule,wherein the next correct nucleotide is identified as a cognate of one ofthe four different base types if ternary complex is detected in thesecond mixture but not the first mixture.

In particular embodiments, a method of nucleic acid detection, caninclude (a) sequentially contacting a primed template nucleic acid withfirst and second mixtures under ternary complex stabilizing conditions,wherein each of the mixtures includes a polymerase and nucleotidecognates for at least two of four different base types in the primedtemplate nucleic acid, wherein the mixtures differ by at least one typeof nucleotide cognate; (b) examining the first and second mixturesseparately to detect ternary complexes; and (c) identifying the nextcorrect nucleotide for the primed template nucleic acid molecule,wherein the next correct nucleotide is identified as a cognate of one ofthe four different base types if ternary complex is not detected in thetwo mixtures.

Also provided is a method of nucleic acid detection that includes stepsof (a) contacting a primed template nucleic acid with a polymerase and afirst mixture of nucleotides under conditions for stabilizing a ternarycomplex at a nucleotide position in the template, wherein the firstmixture includes a nucleotide cognate of a first base type and anucleotide cognate of a second base type; (b) contacting the primedtemplate nucleic acid with a polymerase and a second mixture ofnucleotides under conditions for stabilizing a ternary complex at thenucleotide position in the template, wherein the second mixture includesa nucleotide cognate of the first base type and a nucleotide cognate ofa third base type; (c) contacting the primed template nucleic acid witha polymerase and a third mixture of nucleotides under conditions forstabilizing a ternary complex at the nucleotide position in thetemplate, wherein the third mixture includes a nucleotide cognate of thesecond base type and a nucleotide cognate of a fourth base type; (d)contacting the primed template nucleic acid with a polymerase and afourth mixture of nucleotides under conditions for stabilizing a ternarycomplex at the nucleotide position in the template, wherein the fourthmixture includes a nucleotide cognate of the third base type and anucleotide cognate of the fourth base type; (e) examining products ofsteps (a) through (d) for signals produced by a ternary complex thatincludes the primed template nucleic acid, a polymerase and a nextcorrect nucleotide, wherein signals acquired for the product of step (a)are ambiguous for the first and second base type, wherein signalsacquired for the product of step (b) are ambiguous for the first andthird base type, wherein signals acquired for the product of step (c)are ambiguous for the second and fourth base type, and wherein signalsacquired for the product of step (d) are ambiguous for the third andfourth base type; (f) disambiguating signals acquired in step (e) toidentify a base type that binds the next correct nucleotide.

DETAILED DESCRIPTION

The present disclosure provides improved methods for identifyingnucleotides in a nucleic acid. In some embodiments, multiple nucleotidesare identified via a repetitive sequencing reaction. Various sequencingtechniques can be used to read a template nucleic acid, one position ata time, as a primer is elongated along the template via polymerase basedsynthesis. One such technique, sequencing-by-binding (SBB), is generallybased on repetitive cycles of detecting a stabilized complex that formsat each position along the template (e.g. a ternary complex thatincludes the primed template, a polymerase, and a cognate nucleotide forthe position), under conditions that prevent covalent incorporation ofthe cognate nucleotide into the primer, and then extending the primer toallow detection of the next position along the template. In SBB,detection of the nucleotide at each position of the template occursprior to extension of the primer to the next position.

Generally, SBB is used to distinguish four different nucleotide typesthat can be present at positions along a nucleic acid template. The typeof nucleotide at each position can be distinguished by uniquelylabelling each type of ternary complex (i.e. different types of ternarycomplexes differing in the type of nucleotide it contains) or byseparately delivering the reagents needed to form each type of ternarycomplex. The two configurations provide different advantages whencompared to each other. For example, the former configuration has therelative disadvantage of requiring complex detection hardware havingfour separate detection channels (instead of only one channel which canbe used in the latter configuration). The latter configuration has therelative disadvantage of consuming more time and reagent to accommodatefour different reagent deliveries (instead of the single reagentdelivery possible in the former configuration).

The present disclosure provides alternative reaction configurations andreagent compositions that can minimize or avoid the above disadvantages.In a particular embodiment, an SBB reaction cycle is carried out withonly a subset of the possible nucleotide types that are capable ofserving as cognates for the diversity of base types expected to occur inthe template being sequenced. In this embodiment, the identity of anomitted nucleotide can be imputed. For example, a DNA template can besubjected to an SBB reaction cycle with only three nucleotide types. Thepresence of cognates for the three types of nucleotides can bedistinguished at individual positions of the template according todetection of a stabilized ternary complex that contains the respectivetype of nucleotide, whereas the presence of a cognate of the fourthnucleotide type at a particular position can be imputed based on absenceof any signal for ternary complex formation at the position. Thisembodiment provides the advantage of requiring fewer reagent deliveriesthan would be required if all four nucleotide types were separatelydelivered. Another advantage is that this embodiment requires fewerdetection channels than would be used if unique signals weredistinguished for each of the four nucleotide types. Exemplaryembodiments that utilize imputation are set forth in the Examplessection below in the context of Tables 1, 2 and 5.

In some embodiments, an SBB method is provided that utilizes fewerreagent deliveries and fewer label types than the number of nucleotidetypes that are distinguished. For example, fewer than three reagentdeliveries and fewer than three types of labels can be used in an SBBcycle that, nonetheless, provides information to uniquely identify threedifferent base types in a template nucleic acid. As a more specificexample, two reagent deliveries and two examinations can be carried outin the following order: (1) the first delivery includes reagents capableof forming a first stabilized ternary complex with a first nucleotidetype and a second stabilized ternary complex with a second nucleotidetype (e.g. a dGTP-ternary complex and a dCTP-ternary complex); (2) theproduct of the first delivery is subjected to a first examination; (3)the second delivery includes reagents capable of forming the firststabilized ternary complex with the first nucleotide type and a thirdstabilized ternary complex with a third nucleotide type (e.g.dGTP-ternary complex and a dTTP-ternary complex); and (4) the product ofthe second delivery is subjected to a second examination. The differentternary complexes can be labeled in any of a variety of ways but thelabels need not distinguish one type of ternary complex from another. Inother words, any signals detected in the above examination steps can beambiguous with respect to the type of nucleotide that participated internary complex formation. The results of the second examination can beused to disambiguate the results of the first examination and viceversa. More specifically disambiguation can be achieved by a comparisonwhere: dGTP is identified from the presence of signal in both the firstand second examinations, or dCTP is called from the presence of signalin the first examination and absence of signal in the secondexamination, or dTTP is called from absence of signal in the firstexamination and presence of signal in the second examination.Furthermore, in this example, the absence of signal in both examinationscan be called as if a dATP had been present, even if it was not.Exemplary embodiments that utilize disambiguation are set forth in theExamples section in the context of Tables 2-5.

A further SBB embodiment can employ label switching for one or morenucleotide type in alternate reagent delivery steps. As such, the changein signal that is detected for the same type of stabilized ternarycomplex can be used as a basis for disambiguating the identity of thenucleotide in the ternary complex. More specifically, different types ofstabilized ternary complexes can produce a different combination ofsignal states when multiple different reagent deliveries are compared.The unique combination of signal states across multiple reagentdeliveries provides a signature that uniquely identifies different basetypes in a template nucleic acid. Exemplary embodiments that utilizealternating signal states as a unique signature are set forth in theExamples section in the context of Tables 3, 4, 5, 7 and 8.

As will be apparent from the above example, methods set forth herein canprovide the advantage of reducing the complexity and cost of detectionhardware and can also provide the advantage of reducing cycle time andreagent cost compared to previous SBB methods.

Particular embodiments of the present disclosure provide improvedaccuracy. Using methods set forth herein, ternary complexes can beformed and examined multiple times at a particular position in a primedtemplate. In a sequencing method, this can be achieved by performing acycle that includes multiple reagent deliveries and examination stepsfor a particular position in a primed template prior to advancing to thenext cycle by extending the primer. This can effectively result inrepetitive examinations of a particular cognate nucleotide type at agiven template position. These repetitive examinations can be combinedto provide a more accurate nucleotide call than would be available froma single examination of the particular cognate nucleotide type at thatposition. Moreover, repetitive examinations of this type can providestatistical analysis or variance measures for nucleotides called atindividual positions in a primed template. Such information can in turnbe used to evaluate overall accuracy for a sequence determination.

Repetitive examinations can be achieved by merely repeating steps withina typical SBB cycle. For example, the repeated steps can involvedelivering reagents to form four uniquely labeled types of ternarycomplexes and examining the ternary complexes using detectors thatdistinguish the different types of ternary complexes in a mixture. Asanother example, the repeated steps can involve separately deliveringreagents to form each of four different types of ternary complexes andseparately detecting the product of each delivery. The formerconfiguration has a relative disadvantage of requiring more complexdetection hardware and the latter configuration has a relativedisadvantage of consuming relatively more time and reagents. Inaccordance with disambiguation methods set forth herein, a position in aprimed template can be sequentially treated with different mixtures ofreagents for forming ternary complexes and the resulting mixtures can beexamined. Appropriate selection of nucleotide types across combinatorialmixtures can allow the position to be treated and examined fewer timesor with fewer label types than would be required when merely usingrepetitive delivery of the same reagents. Exemplary embodiments thatutilize disambiguation and provide improved accuracy are set forth inthe Examples section in the context of Tables 3, 9 and 10.

A variety of SBB techniques can be modified in accordance with theteachings set forth herein including, for example, those described incommonly owned U.S. Pat. App. Pub. No. 2017/0022553 A1 or U.S. Pat. App.Ser. Nos. 62/447,319; 62/440,624; or 62/450,397, each of which isincorporated herein by reference.

Furthermore, although methods that employ imputation and disambiguationare exemplified herein with regard to repetitive sequencing reactions,the steps need not be repeated. For example, a genotyping method thatprobes a single nucleotide position in a template nucleic acid viaformation of a stabilized ternary complex can be carried out with only asubset of the possible nucleotide types that would be expected to formcognates with the template being genotyped and the identity of theomitted nucleotide can be imputed. In another example fewer than threereagent deliveries and fewer than three types of labels can be used in agenotyping reaction that, nonetheless, provides information to uniquelyidentify three or four different nucleotide types using disambiguationand/or alternating signal states. Examples of genotyping techniques thatcan be modified to employ imputation and/or disambiguation techniquesset forth herein include those set forth in commonly owned U.S. Pat.App. Ser. No. 62/448,630, which is incorporated herein by reference.

Terms used herein will be understood to take on their ordinary meaningin the relevant art unless specified otherwise. Several terms usedherein and their meanings are set forth below.

As used herein, the term “ambiguous,” when used in reference to asignal, means that the signal apparently has more than one potentialorigin. For example, an ambiguous signal that is acquired in a cycle ofa sequencing reaction may not distinguish between two or more nucleotidetypes that could participate in the cycle to produce the signal. Whenused in reference to a nucleic acid representation (e.g. a nucleic acidsequence), the term “ambiguous” refers to a position in the nucleic acidrepresentation for which two or more nucleotide types are identified ascandidate occupants. An ambiguous position can have, for example, atleast 2, 3 or 4 nucleotide types as candidate occupants. Alternativelyor additionally, an ambiguous position can have at most 4, 3 or 2nucleotide types as candidate occupants.

As used herein, the term “array” refers to a population of moleculesthat are attached to one or more solid-phase substrates such that themolecules at one feature can be distinguished from molecules at otherfeatures. An array can include different molecules that are each locatedat different addressable features on a solid-phase substrate.Alternatively, an array can include separate solid-phase substrates eachfunctioning as a feature that bears a different molecule, wherein thedifferent molecules can be identified according to the locations of thesolid-phase substrates on a surface to which the solid-phase substratesare attached, or according to the locations of the solid-phasesubstrates in a liquid such as a fluid stream. The molecules of thearray can be, for example, nucleotides, nucleic acid primers, nucleicacid templates, primed templates, primed nucleic acid templates, primedtemplate nucleic acid, or nucleic acid enzymes such as polymerases,ligases, exonucleases or combinations thereof.

As used herein, the term “binary complex” refers to an intermolecularassociation between a polymerase and a primed template nucleic acid,exclusive of a nucleotide molecule such as a next correct nucleotide ofthe primed template nucleic acid.

As used herein, the term “blocking moiety,” when used in reference to anucleotide analog, refers to a part of the nucleotide analog thatinhibits or prevents the nucleotide from forming a covalent linkage to anext correct nucleotide (e.g., via the 3′-oxygen of a primer nucleotide)during the incorporation step of a nucleic acid polymerization reaction.The blocking moiety of a “reversible terminator” nucleotide can beremoved from the nucleotide analog, or otherwise modified, to allow the3′-oxygen of the nucleotide to covalently link to a next correctnucleotide. Such a blocking moiety is referred to herein as a“reversible terminator moiety.” Exemplary reversible terminator moietiesare set forth in U.S. Pat. Nos. 7,427,673; 7,414,116; 7,057,026;7,544,794 or 8,034,923; or PCT publications WO 91/06678 or WO 07/123744,each of which is incorporated herein by reference.

As used herein, the term “catalytic metal ion” refers to a metal ionthat facilitates phosphodiester bond formation between the 3′ oxygen ofa nucleic acid (e.g., a primer) and the 5′ phosphate of an incomingnucleotide by a polymerase. A “divalent catalytic metal cation” is acatalytic metal ion having a valence of two. Catalytic metal ions can bepresent at concentrations that stabilize formation of a complex betweena polymerase, nucleotide, and primed template nucleic acid, referred toas non-catalytic concentrations of a metal ion insofar as phosphodiesterbond formation does not occur. Catalytic concentrations of a metal ionrefer to the amount of a metal ion sufficient for polymerases tocatalyze the reaction between the 3′oxygen group of a nucleic acid(e.g., a primer) and the 5′ phosphate group of an incoming nucleotide.

As used herein, the term “comprising” is intended to be open-ended,including not only the recited elements, but further encompassing anyadditional elements.

As used herein, the term “destabilize” means to cause something to beunable to continue existing or working in a particular way.“Destabilizing” a binary complex refers to the process of promotingdissolution or breakdown of the binary complex. “Destabilizing” alsoincludes the process of inhibiting or preventing formation of the binarycomplex.

As used herein the term “determine” can be used to refer to the act ofascertaining, establishing or estimating. A determination can beprobabilistic. For example, a determination can have an apparentlikelihood of at least 50%, 75%, 90%, 95%, 98%, 99%, 99.9% or higher. Insome cases, a determination can have an apparent likelihood of 100%. Anexemplary determination is a maximum likelihood analysis or report.

As used herein, the term “disambiguate,” when used in reference tonucleotide identity, means identifying a single nucleotide type from asignal that is ambiguous for at least two candidate nucleotide types,the single nucleotide type being one of the candidate nucleotide types.

As used herein, the term “each,” when used in reference to a collectionof items, is intended to identify an individual item in the collectionbut does not necessarily refer to every item in the collection.Exceptions can occur if explicit disclosure or context clearly dictatesotherwise.

As used herein, the term “exogenous,” when used in reference to a moietyof a molecule, means a chemical moiety that is not present in a naturalanalog of the molecule. For example, an exogenous label of a nucleotideis a label that is not present on a naturally occurring nucleotide.Similarly, an exogenous label that is present on a polymerase is notfound on the polymerase in its native milieu.

As used herein, the term “extension,” when used in reference to anucleic acid, refers to a process of adding at least one nucleotide tothe 3′ end of the nucleic acid. A nucleotide or oligonucleotide that isadded to a nucleic acid by extension is said to be incorporated into thenucleic acid. Accordingly, the term “incorporating” can be used to referto the process of joining a nucleotide or oligonucleotide to the 3′ endof a nucleic acid by formation of a phosphodiester bond.

As used herein, the term “feature,” when used in reference to an array,means a location in an array where a particular molecule is present. Afeature can contain only a single molecule or it can contain apopulation of several molecules of the same species (i.e. an ensemble ofthe molecules). Alternatively, a feature can include a population ofmolecules that are different species (e.g. a population of ternarycomplexes having different template sequences). Features of an array aretypically discrete. The discrete features can be contiguous or they canhave spaces between each other. An array useful herein can have, forexample, features that are separated by less than 100 micron, 50 micron,10 micron, 5 micron, 1 micron, or 0.5 micron. Alternatively oradditionally, an array can have features that are separated by greaterthan 0.5 micron, 1 micron, 5 micron, 10 micron, 50 micron or 100 micron.The features can each have an area of less than 1 square millimeter, 500square micron, 100 square micron, 25 square micron, 1 square micron orless.

As used herein, the term “identify,” when used in reference to a thing,can be used to refer to recognition of the thing, distinction of thething from at least one other thing or categorization of the thing withat least one other thing. The recognition, distinction or categorizationcan be probabilistic. For example, a thing can be identified with anapparent likelihood of at least 50%, 75%, 90%, 95%, 98%, 99%, 99.9% orhigher. A thing can be identified based on a result of a maximumlikelihood analysis. In some cases, a thing can be identified with anapparent likelihood of 100%.

As used herein, the term “impute,” when used in reference to nucleotideidentity, means inferring the presence of a particular type ofnucleotide at a position in the nucleic acid absent observation of adetectable event attributable to the nucleotide. For example, thepresence of a first nucleotide type at a position in a nucleic acid canbe imputed based on absence of an observed signal for the firstnucleotide type. Optionally, the imputation of the first nucleotidetype's presence at the position can be further influenced by theobservation of signal(s) for one or more other nucleotide type at theposition.

As used herein, the term “label” means a molecule or moiety thereof thatprovides a detectable characteristic. The detectable characteristic canbe, for example, an optical signal such as absorbance of radiation,fluorescence emission, luminescence emission, fluorescence lifetime,fluorescence polarization, or the like; Rayleigh and/or Mie scattering;binding affinity for a ligand or receptor; magnetic properties;electrical properties; charge; mass; radioactivity or the like.Exemplary labels include, without limitation, a fluorophore,luminophore, chromophore, nanoparticle (e.g., gold, silver, carbonnanotubes), heavy atoms, radioactive isotope, mass label, charge label,spin label, receptor, ligand, or the like.

As used herein, the term “mixture,” when used in reference to multiplenucleotide types, means a combination of two or more nucleotide typesthat are simultaneously together, for example, in a liquid or on asurface or as a combination thereof. An exemplary combination is asurface bound reaction component that is in contact with a solutionphase component. A mixture can be distinguished from a chemical compoundin that the two or more different things need not necessarily be infixed proportions, need not lose their individual characteristics,and/or can be separated by physical means.

As used herein, the term “next correct nucleotide” refers to thenucleotide type that will bind and/or incorporate at the 3′ end of aprimer to complement a base in a template strand to which the primer ishybridized. The base in the template strand is referred to as the “nexttemplate nucleotide” and is immediately 5′ of the base in the templatethat is hybridized to the 3′ end of the primer. The next correctnucleotide can be referred to as the “cognate” of the next templatenucleotide and vice versa. Cognate nucleotides that interact with eachother in a ternary complex or in a double stranded nucleic acid are saidto “pair” with each other. A nucleotide having a base that is notcomplementary to the next template base is referred to as an“incorrect”, “mismatch” or “non-cognate” nucleotide. A “nucleotidecognate” of a specified base type (e.g., a nucleotide cognate of a firstbase type, a nucleotide cognate of a seonc base type, a nucleotidecognate of a third base type, or a nucleotide cognate of a fourth basetype) is a nucleotide that is complementary to, and/or capable ofselectively pairing with, the specified base type (e.g., preferentiallypairing with a single specified base type over all other candidate basetypes in a template strand). For example, a nucleotide cognate of afirst base type (e.g., of four possible types) is complementary to,and/or capable of pairing with, a first base type and not a differentbase type (e.g., a second, third, or fourth base type). Likewise, forexample, a nucleotide cognate of a second base type (e.g., of fourpossible types) is complementary to, and/or capable of pairing with, asecond base type not a different base type (e.g., a first, third, orfourth base type). A nucleotide cognate may or may not be the nextcorrect nucleotide. Thus, in some embodiments a nucleotide cognate is anext correct nucleotide (i.e., the nucleotide is capable of pairing tothe base type of the next template nucleotide). In alternativeembodiments, a nucleotide cognate is not a next correct nucleotide(i.e., the nucleotide is not capable of pairing to the base type of thenext template nucleotide, but instead pairs with another type ofnucleotide that is not present at the next template nucleotideposition). In embodiments, a nucleotide cognate of a first, second,third, or fourth base type is capable of pairing with a first, second,third, or fourth base type, respectively, and not one of the three otherbase types. In embodiments, the first second, third, and fourth basetypes are, respectively, A, C, G, T; A, C, T, G; A, G, C, T; A, G, T, C;A, T, C, G; A, T, G, C; C, G, T, A; C, G, A, T; C, T, G, A; C, T, A, G;C, A, G, C; C, A, C, G; G, T, A, C; G, T, C, A; G, A, C, T; G, A, T, C;G, C, T, A; G, C, A, T; C, G, T, A; C, G, A, T; C, T, G, A; C, T, A, G;C, A, G, T; or C, A, T, G, all being commonly used single letter labelsfor DNA base types. In embodiments, the first second, third, and fourthbase types are, respectively, A, C, G, U; A, C, U, G; A, G, C, U; A, G,U, C; A, U, C, G; A, U, G, C; C, G, U, A; C, G, A, U; C, U, G, A; C, U,A, G; C, A, G, C; C, A, C, G; G, U, A, C; G, U, C, A; G, A, C, U; G, A,U, C; G, C, U, A; G, C, A, U; C, G, U, A; C, G, A, U; C, U, G, A; C, U,A, G; C, A, G, U; or C, A, U, G

As used herein, the term “non-catalytic metal ion” refers to a metal ionthat, when in the presence of a polymerase enzyme, does not facilitatephosphodiester bond formation needed for covalent incorporation of anucleotide into a primer. A non-catalytic metal ion may interact with apolymerase, for example, via competitive binding compared to catalyticmetal ions. A “divalent non-catalytic metal ion” is a non-catalyticmetal ion having a valence of two. Examples of divalent non-catalyticmetal ions include, but are not limited to, Ca²⁺, Zn²⁺, Co²⁺, Ni²⁺, andSr²⁺. The trivalent Eu³⁺ and Tb³⁺ ions are non-catalytic metal ionshaving a valence of three.

As used herein, the term “nucleotide” can be used to refer to a nativenucleotide or analog thereof. Examples include, but are not limited to,nucleotide triphosphates (NTPs) such as ribonucleotide triphosphates(rNTPs), deoxyribonucleotide triphosphates (dNTPs), or non-naturalanalogs thereof such as dideoxyribonucleotide triphosphates (ddNTPs) orreversibly terminated nucleotide triphosphates (rtNTPs).

As used herein, the term “polymerase” can be used to refer to a nucleicacid synthesizing enzyme, including but not limited to, DNA polymerase,RNA polymerase, reverse transcriptase, primase and transferase.Typically, the polymerase has one or more active sites at whichnucleotide binding and/or catalysis of nucleotide polymerization mayoccur. The polymerase may catalyze the polymerization of nucleotides tothe 3′ end of the first strand of the double stranded nucleic acidmolecule. For example, a polymerase catalyzes the addition of a nextcorrect nucleotide to the 3′ oxygen group of the first strand of thedouble stranded nucleic acid molecule via a phosphodiester bond, therebycovalently incorporating the nucleotide to the first strand of thedouble stranded nucleic acid molecule. Optionally, a polymerase need notbe capable of nucleotide incorporation under one or more conditions usedin a method set forth herein. For example, a mutant polymerase may becapable of forming a ternary complex but incapable of catalyzingnucleotide incorporation.

As used herein, the term “primed template,” “primed template nucleicacid,” or “primed nucleic acid template” refers to a nucleic acid hybridhaving a double stranded region such that one of the strands has a3′-end that can be extended by a polymerase (e.g., by covalentlyattaching a next correct nucleotide to the 3′-end of the strand),optionally following deblocking of the strand to be extended by thepolymerase. The two strands can be parts of a contiguous nucleic acidmolecule (e.g. a hairpin structure) or the two strands can be separablemolecules that are not covalently attached to each other.

As used herein, the term “primer” means a nucleic acid having a sequencethat binds (e.g., is complementary) to a nucleic acid at or near atemplate sequence. Generally, the primer binds in a configuration thatallows replication of the template, for example, via polymeraseextension of the primer (e.g., the primer may need to be deblocked priorto replication). In embodiments, when hybridized to a template sequence,the primer is capable of binding a polymerase (e.g. thereby allowing forprimer extension). The primer may be of any appropriate length. Theprimer can be a first portion of a nucleic acid molecule that binds to asecond portion of the nucleic acid molecule, the first portion being aprimer sequence and the second portion being a primer binding sequence(e.g. a hairpin primer). Alternatively, the primer can be a firstnucleic acid molecule that binds to a second nucleic acid moleculehaving the template sequence. A primer can consist of DNA, RNA oranalogs thereof.

As used herein, the term “signal” refers to energy or coded informationthat can be selectively observed over other energy or information suchas background energy or information. A signal can have a desired orpredefined characteristic. For example, an optical signal can becharacterized or observed by one or more of intensity, wavelength,energy, frequency, power, luminance or the like. Other signals can bequantified according to characteristics such as voltage, current,electric field strength, magnetic field strength, frequency, power,temperature, etc. An optical signal can be detected at a particularintensity, wavelength, or color; an electrical signal can be detected ata particular frequency, power or field strength; or other signals can bedetected based on characteristics known in the art pertaining tospectroscopy and analytical detection. Absence of signal is understoodto be a signal level of zero or a signal level that is not meaningfullydistinguished from noise.

As used herein, the term “ternary complex” refers to an intermolecularassociation between a polymerase, a double stranded nucleic acid and anucleotide. Typically, the polymerase facilitates interaction between anext correct nucleotide and a template strand of the primed nucleicacid. A next correct nucleotide can interact with the template strandvia Watson-Crick hydrogen bonding. The term “stabilized ternary complex”means a ternary complex having promoted or prolonged existence or aternary complex for which disruption has been inhibited. Generally,stabilization of the ternary complex prevents covalent incorporation ofthe nucleotide component of the ternary complex into the primed nucleicacid component of the ternary complex.

As used herein, the term “type” is used to identify molecules that sharethe same chemical structure. For example, a mixture of nucleotides caninclude several dCTP molecules. The dCTP molecules will be understood tobe the same type as each other, but a different type compared to dATP,dGTP, dTTP etc. Similarly, individual DNA molecules that have the samesequence of nucleotides are the same type, whereas DNA molecules withdifferent sequences are different types. The term “type” can alsoidentify moieties that share the same chemical structure. For example,the cytosine bases in a template nucleic acid will be understood to bethe same type of base as each other independent of their position in thetemplate sequence.

The embodiments set forth below and recited in the claims can beunderstood in view of the above definitions.

The present disclosure provides methods for identifying the base type atone or more positions of a nucleic acid. A reaction can be carried outto form a stabilized ternary complex, between a primed template nucleicacid, a polymerase and only a subset of the possible nucleotide typesthat are candidates for forming cognates with bases in the template. Theidentity of the subset of nucleotides can be identified from detectedsignals whereas a nucleotide that does not participate in the reaction(or at least does not produce a detected signal in the reaction) can beidentified by imputation. It will be understood that, in accordance withWatson-Crick base-pairing rules, the identity of a cognate base at aposition in a nucleic acid can be readily determined from the identityof the type of nucleotide that is present in a stabilized ternarycomplex formed at the position.

In some embodiments, the subset of possible nucleotide types can besimultaneously present in a ternary complex forming reaction.Accordingly, this disclosure provides a method of nucleic aciddetection, that includes the steps of (a) forming a mixture underternary complex stabilizing conditions, wherein the mixture includes aprimed template nucleic acid, a polymerase and nucleotide cognates offirst, second and third base types in the template; (b) examining themixture to determine whether a ternary complex formed; and (c)identifying the next correct nucleotide for the primed template nucleicacid molecule, wherein the next correct nucleotide is identified as acognate of the first, second or third base type if ternary complex isdetected in step (b), and wherein the next correct nucleotide is imputedto be a nucleotide cognate of a fourth base type based on the absence ofa ternary complex in step (b).

Alternatively, different nucleotide types in a subset of candidates canbe serially reacted with a template nucleic acid under conditions toform ternary complex with a polymerase. Accordingly, the presentdisclosure provides a method of nucleic acid detection that includes thesteps of (a) sequentially contacting a primed template nucleic acid withat least two separate mixtures under ternary complex stabilizingconditions, wherein the at least two separate mixtures each include apolymerase and a nucleotide, whereby the sequentially contacting resultsin the primed template nucleic acid being contacted, under the ternarycomplex stabilizing conditions, with nucleotide cognates for first,second and third base types in the template; (b) examining the at leasttwo separate mixtures to determine whether a ternary complex formed; and(c) identifying the next correct nucleotide for the primed templatenucleic acid molecule, wherein the next correct nucleotide is identifiedas a cognate of the first, second or third base type if ternary complexis detected in step (b), and wherein the next correct nucleotide isimputed to be a nucleotide cognate of a fourth base type based on theabsence of a ternary complex in step (b).

Described herein are polymerase-based methods for detecting nucleicacids. Embodiments of the methods exploit the specificity with which apolymerase can form a stabilized ternary complex with a primed templatenucleic acid and a next correct nucleotide. In particular embodiments,the next correct nucleotide is non-covalently bound to the stabilizedternary complex, interacting with the other members of the complexsolely via non-covalent interactions. Useful methods and compositionsfor forming a stabilized ternary complex are set forth in further detailbelow and in commonly owned U.S. Pat. App. Pub. No. 2017/0022553 A1 orU.S. Pat. App. Ser. Nos. 62/447,319; 62/440,624 or 62/450,397, each ofwhich is incorporated herein by reference.

While a ternary complex can form between a polymerase, primed templatenucleic acid and next correct nucleotide in the absence of certaincatalytic metal ions (e.g., Mg²⁺), chemical addition of the nucleotideis inhibited in the absence of the catalytic metal ions. Low ordeficient levels of catalytic metal ions, causes non-covalentsequestration of the next correct nucleotide in a stabilized ternarycomplex. Other methods disclosed herein also can be used to produce astabilized ternary complex.

Optionally, a stabilized ternary complex can be formed when the primerof the primed template nucleic acid includes a blocking moiety (e.g. areversible terminator moiety) that precludes enzymatic incorporation ofan incoming nucleotide into the primer. The interaction can take placein the presence of stabilizers, whereby the polymerase-nucleic acidinteraction is stabilized in the presence of the next correct nucleotide(i.e., stabilizers that stabilize the ternary complex). The primer ofthe primed template nucleic acid optionally can be either an extendibleprimer, or a primer blocked from extension at its 3′-end (e.g., by thepresence of a reversible terminator moiety). The primed template nucleicacid, the polymerase and the cognate nucleotide are capable of forming astabilized ternary complex when the base of the cognate nucleotide iscomplementary to the next base of the primed template nucleic acid(e.g., the next template nucleotide).

As set forth above, conditions that favor or stabilize a ternary complexcan be provided by the presence of a blocking group that precludesenzymatic incorporation of an incoming nucleotide into the primer (e.g.a reversible terminator moiety on the 3′ nucleotide of the primer) orthe absence of a catalytic metal ion. Other useful conditions includethe presence of a ternary complex stabilizing agent such as anon-catalytic ion (e.g., a divalent or trivalent non-catalytic metalion) that inhibits nucleotide incorporation or polymerization.Non-catalytic metal ions include, but are not limited to, calcium,strontium, scandium, titanium, vanadium, chromium, iron, cobalt, nickel,copper, zinc, gallium, germanium, arsenic, selenium, rhodium, europium,and terbium ions. Optionally, conditions that disfavor or destabilizebinary complexes (i.e. complexes between polymerase and primed nucleicacid but lacking cognate nucleotide (e.g., next correct nucleotide)) areprovided by the presence of one or more monovalent cations and/orglutamate anions. As a further option, a polymerase engineered to havereduced catalytic activity or reduced propensity for binary complexformation can be used.

As set forth above, ternary complex stabilization conditions canaccentuate the difference in affinity of polymerase toward primedtemplate nucleic acids in the presence of different nucleotides, forexample, by destabilizing binary complexes. Optionally, the conditionscause differential affinity of the polymerase for the primed templatenucleic acid in the presence of different nucleotides. By way ofexample, the conditions include, but are not limited to, high salt andglutamate ions. For example, the salt may dissolve in aqueous solutionto yield a monovalent cation, such as a monovalent metal cation (e.g.,sodium ion or potassium ion). Optionally, the salt that provides themonovalent cations (e.g., monovalent metal cations) further providesglutamate ions. Optionally, the source of glutamate ions can bepotassium glutamate. In some instances, the concentrations of potassiumglutamate that can be used to alter polymerase affinity of the primedtemplate nucleic acid extend from 10 mM to 1.6 M of potassium glutamate,or any amount in between 10 mM and 1.6 M. As indicated above, high saltrefers to a concentration of salt from 50 to 1,500 mM salt.

It will be understood that options set forth herein for stabilizing aternary complex need not be mutually exclusive and instead can be usedin various combinations. For example, a ternary complex can bestabilized by one or a combination of means including, but not limitedto, crosslinking of the polymerase domains, crosslinking of thepolymerase to the nucleic acid, polymerase mutations that stabilize theternary complex, allosteric inhibition by small molecules, uncompetitiveinhibitors, competitive inhibitors, non-competitive inhibitors, andother means set forth herein.

A stabilized ternary complex can include a native nucleotide, nucleotideanalog or modified nucleotide as desired to suit a particularapplication or configuration of the methods. Optionally, a nucleotideanalog has a nitrogenous base, five-carbon sugar, and phosphate group,wherein any moiety of the nucleotide may be modified, removed and/orreplaced as compared to a native nucleotide. Nucleotide analogs may benon-incorporable nucleotides (i.e. nucleotides that are incapable ofreacting with the 3′ oxygen of a primer to form a covalent linkage).Such nucleotides that are incapable of incorporation include, forexample, monophosphate and diphosphate nucleotides. In another example,the nucleotide may contain modification(s) to the triphosphate groupthat make the nucleotide non-incorporable. Examples of non-incorporablenucleotides may be found in U.S. Pat. No. 7,482,120, which isincorporated by reference herein. In some embodiments, non-incorporablenucleotides may be subsequently modified to become incorporable.Non-incorporable nucleotide analogs include, but are not limited to,alpha-phosphate modified nucleotides, alpha-beta nucleotide analogs,beta-phosphate modified nucleotides, beta-gamma nucleotide analogs,gamma-phosphate modified nucleotides, or caged nucleotides. Examples ofnucleotide analogs are described in U.S. Pat. No. 8,071,755, which isincorporated by reference herein.

Nucleotide analogs that participate in stabilized ternary complexes caninclude terminators that reversibly prevent nucleotide incorporation atthe 3′-end of the primer after the analog has been incorporated. Forexample, U.S. Pat. No. 7,544,794 and U.S. Pat. No. 8,034,923 (thedisclosures of these patents are incorporated herein by reference)describe reversible terminators in which the 3′-OH group is replaced bya 3′-ONH2 moiety. Another type of reversible terminator is linked to thenitrogenous base of a nucleotide as set forth, for example, in U.S. Pat.No. 8,808,989 (the disclosure of which is incorporated herein byreference). Other reversible terminators that similarly can be used inconnection with the methods described herein include those described inreferences cited previously herein or in U.S. Pat. No. 7,956,171, U.S.Pat. No. 8,071,755, and U.S. Pat. No. 9,399,798 (the disclosures ofthese U.S. patents are incorporated herein by reference). In certainembodiments, a reversible blocking moiety can be removed from a primer,in a process known as “deblocking,” allowing for subsequent nucleotideincorporation. Compositions and methods for deblocking are set forth inreferences cited herein in the context of reversible terminators.

Alternatively, nucleotide analogs irreversibly prevent nucleotideincorporation at the 3′-end of the primer to which they have beenincorporated. Irreversible nucleotide analogs include2′,3′-dideoxynucleotides (ddNTPs such as ddGTP, ddATP, ddTTP, ddCTP).Dideoxynucleotides lack the 3′-OH group of dNTPs that would otherwiseparticipate in polymerase-mediated primer extension. Irreversiblyterminated nucleotides can be particularly useful for genotypingapplications.

In some embodiments, a nucleotide that participates in forming a ternarycomplex can include an exogenous label. For example, an exogenouslylabeled nucleotide can include a reversible or irreversible terminatormoiety, an exogenously labeled nucleotide can be non-incorporable, anexogenously labeled nucleotide can lack terminator moieties, anexogenously labeled nucleotide can be incorporable or an exogenouslylabeled nucleotide can be both incorporable and non-terminated.Exogenously labelled nucleotides can be particularly useful when used toform a stabilized ternary complex with a non-labelled polymerase.Alternatively, an exogenous label on a nucleotide can provide onepartner in a fluorescence resonance energy transfer (FRET) pair and anexogenous label on a polymerase can provide the second partner of thepair. As such, FRET detection can be used to identify a stabilizedternary complex that includes both partners. Alternatively, a nucleotidethat participates in forming a ternary complex can lack exogenous labels(i.e. the nucleotide can be “non-labeled”). For example, a non-labelednucleotide can include a reversible or irreversible terminator moiety, anon-labeled nucleotide can be non-incorporable, a non-labeled nucleotidecan lack terminator moieties, a non-labeled nucleotide can beincorporable or a non-labeled labeled nucleotide can be bothincorporable and non-terminated. Non-labelled nucleotides can be usefulwhen a label on a polymerase is used to detect a stabilized ternarycomplex. Non-labelled nucleotides can also be useful in an extensionstep of an SBB method. It will be understood that absence of a moiety orfunction for a nucleotide refers to the nucleotide having no suchfunction or moiety. However, it will also be understood that one or moreof the functions or moieties set forth herein for a nucleotide, oranalog thereof, or otherwise known in the art for a nucleotide, oranalog thereof, can be specifically omitted in a method or compositionset forth herein.

Optionally, a nucleotide (e.g. a native nucleotide or nucleotide analog)is present in a mixture during formation of a stabilized ternarycomplex. For example, at least 1, 2, 3, 4 or more nucleotide types canbe present. Alternatively or additionally, at most 4, 3, 2, or 1nucleotide types can be present. Similarly, one or more nucleotide typesthat are present can be complementary to at least 1, 2, 3 or 4 basetypes in a template nucleic acid. Alternatively or additionally, one ormore nucleotide types that are present can be complementary to at most4, 3, 2, or 1 base types in a template nucleic acid.

Any nucleotide modification that stabilizes a polymerase in a ternarycomplex may be used in the methods disclosed herein. The nucleotide maybe bound permanently or transiently to a polymerase. Optionally, anucleotide analog is fused to a polymerase, for example, via a covalentlinker. Optionally, a plurality of nucleotide analogs are fused to aplurality of polymerases, wherein each nucleotide analog is fused to adifferent polymerase. Optionally, a nucleotide that is present in astabilized ternary complex is not the means by which the ternary complexis stabilized. Accordingly, any of a variety of other ternary complexstabilization methods may be combined in a reaction utilizing anucleotide analog.

In particular embodiments, the primer strand of a primed templatenucleic acid molecule that is present in a stabilized ternary complex ischemically unchanged by the polymerase that is present during one ormore steps of a method set forth herein. For example, the primer neednot be extended by formation of a new phosphodiester bond, nor shortenedby nucleolytic degradation during a step for forming a stabilizedternary complex, nor during a step for examining the stabilized ternarycomplex.

Any of a variety of polymerases can be used to form a stabilized ternarycomplex in a method set forth herein. Polymerases that may be usedinclude naturally occurring polymerases and modified variations thereof,including, but not limited to, mutants, recombinants, fusions, geneticmodifications, chemical modifications, synthetics, and analogs.Naturally occurring polymerases and modified variations thereof are notlimited to polymerases that have the ability to catalyze apolymerization reaction. Optionally, the naturally occurring and/ormodified variations thereof have the ability to catalyze apolymerization reaction in at least one condition that is not usedduring formation or examination of a stabilized ternary complex.Optionally, the naturally-occurring and/or modified variations thatparticipate in stabilized ternary complexes have modified properties,for example, enhanced binding affinity to nucleic acids, reduced bindingaffinity to nucleic acids, enhanced binding affinity to nucleotides,reduced binding affinity to nucleotides, enhanced specificity for nextcorrect nucleotides, reduced specificity for next correct nucleotides,reduced catalysis rates, catalytic inactivity etc. Mutant polymerasesinclude, for example, polymerases wherein one or more amino acids arereplaced with other amino acids, or insertions or deletions of one ormore amino acids.

Modified polymerases include polymerases that contain an exogenous labelmoiety (e.g., an exogenous fluorophore), which can be used to detect thepolymerase. Optionally, the label moiety can be attached after thepolymerase has been at least partially purified using protein isolationtechniques. For example, the exogenous label moiety can be chemicallylinked to the polymerase using a free sulfhydryl or a free amine moietyof the polymerase. This can involve chemical linkage to the polymerasethrough the side chain of a cysteine residue, or through the free aminogroup of the N-terminus. An exogenous label moiety can also be attachedto a polymerase via protein fusion. Exemplary label moieties that can beattached via protein fusion include, for example, green fluorescentprotein (GFP), phycobiliproteins (e.g. phycocyanin and phycoerythrin) orwavelength-shifted variants of GFP or phycobiliproteins. In someembodiments, an exogenous label on a polymerase can function as a memberof a FRET pair. The other member of the fret pair can be an exogenouslabel that is attached to a nucleotide that binds to the polymerase in astabilized ternary complex. As such, the stabilized ternary complex canbe detected or identified via FRET.

Alternatively, a polymerase that participates in a stabilized ternarycomplex need not be attached to an exogenous label. For example, thepolymerase need not be covalently attached to an exogenous label.Instead, the polymerase can lack any label until it associates with alabeled nucleotide and/or labeled nucleic acid (e.g. labeled primerand/or labeled template).

A ternary complex that is made or used in accordance with the presentdisclosure may optionally include one or more exogenous label(s). Thelabel can be attached to a component of the ternary complex (e.g.attached to the polymerase, template nucleic acid, primer and/or cognatenucleotide) prior to formation of the ternary complex. Exemplaryattachments include covalent attachments or non-covalent attachmentssuch as those set forth herein, in references cited herein or known inthe art. In some embodiments, a labeled component is delivered insolution to a solid support that is attached to an unlabeled component,whereby the label is recruited to the solid support by virtue of forminga stabilized ternary complex. As such, the support-attached componentcan be detected or identified based on observation of the recruitedlabel. Whether used in solution phase or on a solid support, exogenouslabels can be useful for detecting a stabilized ternary complex or anindividual component thereof, during an examination step. An exogenouslabel can remain attached to a component after the component dissociatesfrom other components that had formed a stabilized ternary complex.Exemplary labels, methods for attaching labels and methods for usinglabeled components are set forth in commonly owned U.S. Pat. App. Pub.No. 2017/0022553 A1 or U.S. Pat. App. Ser. Nos. 62/447,319; 62/440,624or 62/450,397, each of which is incorporated herein by reference.

Examples of useful exogenous labels include, but are not limited to,radiolabel moieties, luminophore moieties, fluorophore moieties, quantumdot moieties, chromophore moieties, enzyme moieties, electromagneticspin labeled moieties, nanoparticle light scattering moieties, and anyof a variety of other signal generating moieties known in the art.Suitable enzyme moieties include, for example, horseradish peroxidase,alkaline phosphatase, beta-galactosidase, or acetylcholinesterase.Exemplary fluorophore moieties include, but are not limited toumbelliferone, fluorescein, isothiocyanate, rhodamine, tetramethylrhodamine, eosin, green fluorescent protein, erythrosin, coumarin,methyl coumarin, pyrene, malachite green, stilbene, lucifer Yellow™,Cascade Blue™, Texas Red™, dansyl chloride, phycoerythrin, phycocyanin,fluorescent lanthanide complexes such as those including Europium andTerbium, Cy3, Cy5, and others known in the art such as those describedin Principles of Fluorescence Spectroscopy, Joseph R. Lakowicz (Editor),Plenum Pub Corp, 2nd edition (July 1999) and the 6th Edition ofMolecular Probes Handbook by Richard P. Hoagland.

A secondary label can be used in a method of the present disclosure. Asecondary label is a binding moiety that can bind specifically to alabeled partner moiety. For example, a ligand moiety can be attached toa polymerase, nucleic acid or nucleotide to allow detection via specificaffinity for labeled receptor. Exemplary pairs of binding moieties thatcan be used include, without limitation, antigen and immunoglobulin oractive fragments thereof, such as FAbs; immunoglobulin andimmunoglobulin (or active fragments, respectively); avidin and biotin,or analogs thereof having specificity for avidin; streptavidin andbiotin, or analogs thereof having specificity for streptavidin; orcarbohydrates and lectins.

In some embodiments, the secondary label can be a chemically modifiablemoiety. In this embodiment, labels having reactive functional groups canbe incorporated into a stabilized ternary complex. Subsequently, thefunctional group can be covalently reacted with a primary label moiety.Suitable functional groups include, but are not limited to, aminogroups, carboxy groups, maleimide groups, oxo groups and thiol groups.

In alternative embodiments, a ternary complex can lack exogenous labels.For example, a ternary complex and all components participating in theternary complex (e.g. polymerase, template nucleic acid, primer and/orcognate nucleotide) can lack one, several or all of the exogenous labelsdescribed herein or in the above-incorporated references. In suchembodiments, ternary complexes can be detected based on intrinsicproperties of the stabilized ternary complex, such as mass, charge,intrinsic optical properties or the like. Exemplary methods fordetecting non-labeled ternary complexes are set forth in commonly ownedU.S. Pat. App. Pub. No. 2017/0022553 A1 PCT App. Ser. No.PCT/US16/68916, or U.S. Pat. App. Ser. No. 62/375,379, each of which isincorporated herein by reference.

A method of the present disclosure can include an examination step.Generally, detection can be achieved in an examination step by methodsthat perceive a property that is intrinsic to a ternary complex or alabel moiety attached thereto. Exemplary properties upon which detectioncan be based include, but are not limited to, mass, electricalconductivity, energy absorbance, fluorescence or the like. Detection ofluminescence can be carried out using methods known in the artpertaining to nucleic acid arrays. A luminophore can be detected basedon any of a variety of luminescence properties including, for example,emission wavelength, excitation wavelength, fluorescence resonanceenergy transfer (FRET) intensity, quenching, anisotropy or lifetime.Other detection techniques that can be used in a method set forth hereininclude, for example, mass spectrometry which can be used to perceivemass; surface plasmon resonance which can be used to perceive binding toa surface; absorbance which can be used to perceive the wavelength ofthe energy a label absorbs; calorimetry which can be used to perceivechanges in temperature due to presence of a label; electricalconductance or impedance which can be used to perceive electricalproperties of a label, or other known analytic techniques. Examples ofreagents and conditions that can be used to create, manipulate anddetect stabilized ternary complexes include, for example, those setforth in commonly owned U.S. Pat. App. Pub. No. 2017/0022553 A1; PCTApp. Ser. No. PCT/US16/68916; or U.S. Pat. App. Ser. Nos. 62/447,319;62/440,624 or 62/450,397, each of which is incorporated herein byreference.

In particular embodiments, signal is not detected for stabilized ternarycomplex formed with a particular nucleotide type and the identity of thenucleotide is imputed. In such embodiments, a primed template nucleicacid need not be contacted with that particular nucleotide during any orall examination or detection steps of the method. Alternatively, theparticular nucleotide can be present during an examination step, butundetectable under the conditions employed. For example, the nucleotidemay form a stabilized ternary complex that is not detectable. The lackof detectability may derive from absence of an exogenous label on thenucleotide or on the polymerase that it binds to in the stabilizedternary complex, or the lack of detectability may derive from use of adetection condition that is not configured to detect a label that ispresent on the nucleotide or on the polymerase that it binds to in thestabilized ternary complex. As set forth in further detail below, one ormore nucleotide types that are not present during an examination stepcan nevertheless be provided during an extension step.

It may be advantageous to include all four different types ofnucleotides in a mixture even though the mixture will be examined foronly a subset of ternary complex types. For example, the mixture caninclude nucleotides having all four base types, wherein a first subsetof the nucleotides (e.g. A and G) have a label that is detected and asecond subset of the nucleotides (e.g. C and T) do not have the labelthat is detected. The presence of all four nucleotide types in a mixturecan help prevent formation of ternary complexes having non-cognatenucleotides because correct nucleotides will be present to out competeincorrect nucleotides in the binding mixture. Thus, the presence of allfour nucleotide types can favor formation of ternary complexes havingcorrectly bound cognate nucleotides to improve accuracy of sequencingresults.

Particular embodiments of the methods set forth herein include a step offorming a mixture that includes several components. For example, amixture can be formed between a primed template nucleic acid, apolymerase and one or more nucleotide types. The components of themixture can be delivered to a vessel in any desired order or they can bedelivered simultaneously. Furthermore, some of the components can bemixed with each other to form a first mixture that is subsequentlycontacted with other components to form a more complex mixture. Takingas an example, a step of forming a mixture that includes a primedtemplate nucleic acid, a polymerase and a plurality of differentnucleotide types, it will be understood that the different nucleotidetypes in the plurality can be contacted with each other prior to beingcontacted with the primed template nucleic acid. Alternatively, two ormore of the nucleotide types can be delivered separately to the primedtemplate nucleic acid and/or the polymerase. As such, a first nucleotidetype can be contacted with the primed template nucleic acid prior tobeing contacted with a second nucleotide type. Alternatively oradditionally, the first nucleotide type can be contacted with thepolymerase prior to being contacted with a second nucleotide type.

Some embodiments of the methods set forth herein utilize two or moredistinguishable signals to distinguish stabilized ternary complexes fromeach other and/or to distinguish one base type in a template nucleicacid from another base type. For example, two or more luminophores canbe distinguished from each other based on unique optical properties suchas unique wavelength for excitation or unique wavelength of emission. Inparticular embodiments, a method can distinguish different stabilizedternary complexes based on differences in luminescence intensity. Forexample, a first ternary complex can be detected in a condition where itemits less intensity than a second ternary complex. Such intensityscaling (sometimes called ‘grey scaling’) can exploit anydistinguishable intensity difference. Exemplary difference include aparticular stabilized ternary complex having an intensity that is 10%,25%, 33%, 50%, 66%, or 75% compared to the intensity of anotherstabilized ternary complex that is to be detected.

Intensity differences can be achieved using different luminophores eachhaving a different extinction coefficient (i.e. resulting in differentexcitation properties) and/or different luminescence quantum yield (i.e.resulting in different emission properties). Alternatively, the sameluminophore type can be used but can be present in different amounts.For example, all members of a first population of ternary complexes canbe labeled with a particular luminophore, whereas a second populationhas only half of its members labeled with the luminophore. In thisexample, the second population would be expected to produce half thesignal of the first population. The second population can be produced,for example, by using a mixture of labeled nucleotides and unlabelednucleotides (in contrast to the first population containing primarilylabeled nucleotides). Similarly, the second population can be produced,for example, by using a mixture of labeled polymerases and unlabeledpolymerases (in contrast to the first population containing primarilylabeled polymerases). In an alternative labeling scheme, a firstpopulation of ternary complexes can include polymerase molecules thathave multiple labels that produce a particular luminescent signal and asecond population of ternary complexes can include polymerase moleculesthat each have only one of the labels that produces the luminescentsignal.

The present disclosure provides a method of nucleic acid detection thatincludes the steps of (a) contacting a primed template nucleic acid witha polymerase and a first mixture of nucleotides under ternary complexstabilizing conditions, wherein the first mixture includes a nucleotidecognate of a first base type and a nucleotide cognate of a second basetype; (b) contacting the primed template nucleic acid with a polymeraseand a second mixture of nucleotides under ternary complex stabilizingconditions, wherein the second mixture includes a nucleotide cognate ofthe first base type and a nucleotide cognate of a third base type; (c)examining products of steps (a) and (b) for signals produced by aternary complex that includes the primed template nucleic acid, apolymerase and a next correct nucleotide, wherein signals acquired forthe product of step (a) are ambiguous for the first and second basetype, and wherein signals acquired for the product of step (b) areambiguous for the first and third base type; (d) disambiguating signalsacquired in step (c) to identify a base type that binds the next correctnucleotide. Optionally, to achieve disambiguation (i) the first basetype is correlated with presence of signals for the product of step (a)and presence of signals for the product of step (b), (ii) the secondbase type is correlated with presence of signals for the product of step(a) and absence of signals for the product of step (b), and (iii) thethird base type is correlated with absence of signals for the product ofstep (a) and presence of signals for the product of step (b).

Also provided is a method of nucleic acid detection that includes thesteps of (a) contacting a primed template nucleic acid with a firstmixture including a polymerase, a nucleotide cognate of a first basetype in the template and a nucleotide cognate of a second base type inthe template, wherein the contact occurs in a binding reaction that (i)stabilizes ternary complexes including the primed template nucleic acid,the polymerase and a next correct nucleotide, and (ii) preventsincorporation of the next correct nucleotide into the primer; (b)examining the binding reaction to determine whether a ternary complexformed; (c) subjecting the primed template nucleic acid to a repetitionof steps (a) and (b), wherein the first mixture is replaced with asecond mixture, the second mixture including a polymerase, a nucleotidecognate of the first base type in the template and a nucleotide cognateof a third base type in the template; and (d) identifying the nextcorrect nucleotide for the primed template nucleic acid using theexamination of the binding reactions, or a product thereof, wherein (i)the next correct nucleotide is identified as a cognate of the first basetype if ternary complex is detected in step (b) and detected in therepetition of step (b), (ii) the next correct nucleotide is identifiedas a cognate of the second base type if ternary complex is detected instep (b) and undetected in the repetition of step (b), and (iii) thenext correct nucleotide is identified as a cognate of the third basetype if ternary complex is undetected in step (b) and detected in therepetition of step (b).

In particular embodiments, a primed template nucleic acid can becontacted with two or more mixtures under ternary complex stabilizingconditions. The primed template nucleic acid can be sequentiallycontacted with the mixtures. For example, a primed template nucleic acidcan be contacted with a polymerase and nucleotides under ternary complexstabilization conditions and then the polymerase can be replaced withanother polymerase under ternary complex stabilization conditions.Alternatively or additionally, one or more of the nucleotides can bereplaced with one or more other nucleotides under ternary complexstabilizing conditions. In some embodiments, the polymerase and all ofthe nucleotides of the first mixture are replaced with anotherpolymerase and other nucleotides. In an alternative embodiment, two ormore mixtures can be in simultaneous contact with a primed templatenucleic acid under ternary complex stabilizing conditions.

In many embodiments, a primed template nucleic acid can be contactedwith two or more mixtures under ternary complex stabilizing conditions,wherein the first mixture is formed with the primed template nucleicacid and at least one nucleotide type that differs from at least onenucleotide type present in the second mixture. In such cases, the sametype of polymerase can be present in both the first and second mixtures,either because the polymerase is not removed from the first mixture whenthe second mixture is formed or because the polymerase is removed fromthe first mixture and replaced with a polymerase of the same type. It isalso possible to replace the polymerase of a first mixture with apolymerase of a different type when forming the second mixture.Polymerase replacement can be used, for example, to exploit differentproperties or activities. For example, a first mixture can include afirst nucleotide type and a first polymerase having relatively highaffinity or specificity for the first nucleotide type, and the secondmixture can have a second nucleotide type and a second polymerase havingrelatively high affinity or specificity for the second nucleotide type.In this example, the first polymerase can have higher affinity orspecificity for the first nucleotide compared to the second nucleotidetype. Alternatively or additionally, the second polymerase can havehigher affinity or specificity for the second nucleotide type comparedto the first nucleotide type. In another example, it may be desirablefor the second mixture to include a polymerase that is more convenientlyconverted from a ternary complex stabilized state to a primer extendingstate (as compared to the polymerase used to form the first ternarycomplex).

In embodiments where a primed template nucleic acid is sequentiallycontacted with two or more polymerase-nucleotide mixtures under ternarycomplex stabilizing conditions, examination can be carried out aftereach sequential contact. For example, the primed template nucleic acidcan be contacted with a polymerase and nucleotide to form a firstmixture, then the first mixture can be examined for ternary complexformation, then the primed template nucleic acid can be contacted with apolymerase and a nucleotide to form a second mixture, and then thesecond mixture can be examined for ternary complex formation.Alternatively, two or more mixtures can be formed prior to carrying outan examination step. As such, examination need not intervene two or moresequential steps of contacting a primed template nucleic acid withreagents for forming stabilized ternary complexes.

One or more wash steps can be useful for separating a primed templatenucleic acid from other reagents that were contacted with the primedtemplate nucleic acid under ternary complex stabilizing conditions. Sucha wash can remove one or more reagents from interfering with examinationof a mixture or from contaminating a second mixture that is to be formedon a substrate (or in a vessel) that had previously been in contact withthe first mixture. For example, a primed template nucleic acid can becontacted with a polymerase and at least one nucleotide type to form afirst mixture under ternary complex stabilizing conditions, and thefirst mixture can be examined. Optionally, a wash can be carried outprior to examination to remove reagents that are not participating information of a stabilized ternary complex. Alternatively oradditionally, a wash can be carried out after the examination step toremove one or more component of the first mixture from the primedtemplate nucleic acid. Then the primed template nucleic acid can becontacted with a polymerase and at least one other nucleotide to form asecond mixture under ternary complex stabilizing conditions, and thesecond mixture can be examined for ternary complex formation. As before,an optional wash can be carried out prior to the second examination toremove reagents that are not participating in formation of a stabilizedternary complex.

A method set forth herein, can include a step of examining a mixturethat includes a primed template nucleic acid, polymerase, nucleotidecognate of a first base type in the template and nucleotide cognate of asecond base type in the template, wherein signals acquired from themixture are ambiguous for the first and second base type. The ambiguitycan arise, for example, when signals are acquired from an exogenouslabel attached to the polymerase, such that the signals do notdistinguish which nucleotide type is present in a stabilized ternarycomplex that is detected via the label. Ambiguity can arise when thenucleotides in the mixture do not have exogenous labels or when thedifferent nucleotides do not have unique labels. For example, when twoor more nucleotides in a mixture are attached to the same type ofexogenous label (or to different exogenous labels that produce anoverlapping signal) a signal arising from the mixture, althoughindicating presence of a ternary complex, may not provide adequateinformation to distinguish a ternary complex having one of thenucleotides from a ternary complex having the other nucleotide. However,in some embodiments that produce ambiguous signal, the identity of thenucleotides can be disambiguated by examining the same primed templatenucleic acid in the presence of a second mixture under ternary complexstabilizing conditions. Specifically, the second mixture can lack one ofthe nucleotide types that was in the first mixture. The nucleotide typethat was present in both mixtures can be identified based on the factthat signal was detected in both mixtures, whereas a nucleotide typethat was present in a first mixture and not the second mixture can beidentified based on presence of signal in the first mixture and lack ofsignal in the second mixture. Several specific examples, that utilizedisambiguation are set forth in the Examples below (see Tables 2-5).

An advantage of the disambiguation methods set forth herein is that thenumber of different nucleotide types that are uniquely identified cansurpass the number of unique signals detected (or the number of labelsused). For example, two or more nucleotide types can be distinguished ina method set forth herein based on detection of a signal that is commonto both. By way of further example, signals from a first mixture havinga primed template nucleic acid, polymerase and two or more nucleotidescan be acquired by a detector that is also used to detect signals from asecond mixture having the primed template nucleic acid, a polymerase andtwo or more nucleotides. In this example, the first mixture can includea nucleotide cognate of a first base type and a nucleotide cognate of asecond base type, whereas the second mixture can include a nucleotidecognate of the first base type and a nucleotide cognate of a third basetype. As a further option in this example, signals acquired for thefirst mixture can be ambiguous for the first and second base type, andsignals acquired for the second mixture can be ambiguous for the firstand third base type.

As exemplified by several embodiments set forth herein, three base typesat a particular position of a primed template nucleic acid can bedistinguished using as few as two binding reactions and as few as onetype of label. In such embodiments, the first binding reaction includesa first and second nucleotide type, and the second binding reactionincludes the first nucleotide type and a third nucleotide type. The endresult is that the first nucleotide type is determined when signal isobserved from stabilized ternary complexes formed in both reactions, thesecond nucleotide type is determined when signal is observed for aternary complex formed in the first reaction only, and the thirdnucleotide type is determined when signal is observed for a ternarycomplex formed in the second reaction only. In this example, the fourthnucleotide type need not participate in a binding reaction with theprimed template or, even if the fourth nucleotide is present it need notever be detected. Rather, the fourth nucleotide can be identified byimputation. More specifically, in the case where the template is anaturally occurring nucleic acid (e.g. genomic DNA or mRNA) it is knownthat only four types of nucleotides will be present in the template andthe absence of signal in both binding reactions can be used to imputethat the fourth base type was present at the template position underexamination.

As an alternative to imputing the fourth nucleotide type in the aboveexample, a third binding reaction can be performed using the fourth basetype and a stabilized ternary complex that includes the fourth base typecan be detected. This alternative provides the advantage of confirmingthe results of the first two binding reactions based on observations ofconsistent results (e.g. the fourth nucleotide type is observed only inthe third binding reaction) or identifying a potential error wheninconsistent results are obtained (e.g. the fourth nucleotide type isobserved in the third binding reaction and in the first or secondbinding reaction). This alternative can still provide the advantage ofrequiring fewer reagent delivery steps than the number of nucleotidetypes distinguished (i.e. four nucleotide types are distinguished from 3reagent delivery steps) using as few as one label type.

As demonstrated by the examples set forth herein, four base types can bedistinguished at a particular position of a primed template nucleic acidby examining products of a first binding reaction that includesdetectable ternary complexes having first and second nucleotide typesbut lacks detectable ternary complexes having third and fourthnucleotide types, and examining products of a second binding reactionthat includes detectable ternary complexes having the first nucleotidetype and the third nucleotide type but lacks detectable ternarycomplexes having second and fourth nucleotide types. A third bindingreaction need not be performed nor examined. In this case, speed can beimproved and/or costs reduced by employing imputation to identify thefourth nucleotide type. However, if desired for example to improveaccuracy of sequencing, examination can be carried out for a thirdbinding reaction that includes detectable ternary complexes having onlythe fourth nucleotide type, or that includes detectable ternarycomplexes having the fourth nucleotide type along with detectableternary complexes having one other nucleotide type (but no more than oneother nucleotide type).

Generally, accuracy can be improved by repeating reagent delivery andexamination steps of a method set forth herein when evaluating aparticular position in a primed template nucleic acid. In this way, theposition can be tested multiple times for its ability to form a ternarycomplex with a particular type of nucleotide. Indeed, all four types ofnucleotides can be evaluated repetitively for the ability to formternary complex at a particular position in a primed template. In asequencing by binding embodiment, evaluation can proceed at a subsequentposition of the primed template by performing a primer extension stepfollowing the repeated examination steps.

Accordingly, this disclosure provides a method of nucleic acid detectionthat includes steps of (a) sequentially contacting a primed templatenucleic acid with at least four separate mixtures under ternary complexstabilizing conditions, wherein each of the mixtures includes apolymerase and nucleotide cognates for at least two of four differentbase types in the primed template nucleic acid; (b) examining the atleast four separate mixtures to detect ternary complexes; and (c)identifying the next correct nucleotide for the primed template nucleicacid molecule, wherein the next correct nucleotide is identified as acognate of one of the four different base types if ternary complex isdetected in at least two of the mixtures.

In an aspect is provided a method of nucleic acid detection thatincludes steps of (a) sequentially contacting a primed template nucleicacid with at least four separate mixtures under ternary complexstabilizing conditions, wherein each of the mixtures includes apolymerase and nucleotide cognates for at least two of four differentbase types in the primed template nucleic acid and each of the mixturesincludes a different combination of nucleotide cognates for at least twoof four different base types in the primed template nucleic acid; (b)examining the at least four separate mixtures to detect ternarycomplexes; and (c) identifying the next correct nucleotide for theprimed template nucleic acid molecule, wherein the next correctnucleotide is identified as a cognate of one of the four different basetypes if ternary complex is detected in at least two of the mixtures.

Also provided is a method of nucleic acid detection that includes stepsof (a) contacting a primed template nucleic acid with a polymerase and afirst mixture of nucleotides under conditions for stabilizing a ternarycomplex at a nucleotide position in the template, wherein the firstmixture includes a nucleotide cognate of a first base type and anucleotide cognate of a second base type; (b) contacting the primedtemplate nucleic acid with a polymerase and a second mixture ofnucleotides under conditions for stabilizing a ternary complex at thenucleotide position in the template, wherein the second mixture includesa nucleotide cognate of the first base type and a nucleotide cognate ofa third base type; (c) contacting the primed template nucleic acid witha polymerase and a third mixture of nucleotides under conditions forstabilizing a ternary complex at the nucleotide position in thetemplate, wherein the third mixture includes a nucleotide cognate of thesecond base type and a nucleotide cognate of a fourth base type; (d)contacting the primed template nucleic acid with a polymerase and afourth mixture of nucleotides under conditions for stabilizing a ternarycomplex at the nucleotide position in the template, wherein the fourthmixture includes a nucleotide cognate of the third base type and anucleotide cognate of the fourth base type; (e) examining products ofsteps (a) through (d) for signals produced by a ternary complex thatincludes the primed template nucleic acid, a polymerase and a nextcorrect nucleotide, wherein signals acquired for the product of step (a)are ambiguous for the first and second base type, wherein signalsacquired for the product of step (b) are ambiguous for the first andthird base type, wherein signals acquired for the product of step (c)are ambiguous for the second and fourth base type, and wherein signalsacquired for the product of step (d) are ambiguous for the third andfourth base type; (f) disambiguating signals acquired in step (e) toidentify a base type that binds the next correct nucleotide.

In particular embodiments, the steps of a nucleic acid detection methodset forth herein can be repeated to interrogate several differentpositions in a template nucleic acid. In some cases, a series ofsequential positions along the template can be interrogated.Accordingly, this disclosure provides a method for sequencing a nucleicacid that includes the steps of (a) forming a mixture under ternarycomplex stabilizing conditions, wherein the mixture includes a primedtemplate nucleic acid, a polymerase and nucleotide cognates of first,second and third base types in the template; (b) examining the mixtureto determine whether a ternary complex formed; (c) identifying the nextcorrect nucleotide for the primed template nucleic acid molecule,wherein the next correct nucleotide is identified as a cognate of thefirst, second or third base type if ternary complex is detected in step(b), and wherein the next correct nucleotide is imputed to be anucleotide cognate of a fourth base type based on the absence of aternary complex in step (b); (d) adding a next correct nucleotide to theprimer of the primed template nucleic acid after step (b), therebyproducing an extended primer; and (e) repeating steps (a) through (d)for the primed template nucleic acid that comprises the extended primer.

Also provided by this disclosure is a method for sequencing a nucleicacid that includes the steps of (a) sequentially contacting a primedtemplate nucleic acid with at least two separate mixtures under ternarycomplex stabilizing conditions, wherein the at least two separatemixtures each include a polymerase and a nucleotide, whereby thesequentially contacting results in the primed template nucleic acidbeing contacted, under the ternary complex stabilizing conditions, withnucleotide cognates for first, second and third base types in thetemplate; (b) examining the at least two separate mixtures to determinewhether a ternary complex formed; and (c) identifying the next correctnucleotide for the primed template nucleic acid molecule, wherein thenext correct nucleotide is identified as a cognate of the first, secondor third base type if ternary complex is detected in step (b), andwherein the next correct nucleotide is imputed to be a nucleotidecognate of a fourth base type based on the absence of a ternary complexin step (b); (d) adding a next correct nucleotide to the primer of theprimed template nucleic acid after step (b), thereby producing anextended primer; and (e) repeating steps (a) through (d) for the primedtemplate nucleic acid that comprises the extended primer.

In a further embodiment, a method of nucleic acid sequencing can includethe steps of (a) contacting a primed template nucleic acid with apolymerase and a first mixture of nucleotides under ternary complexstabilizing conditions, wherein the first mixture includes a nucleotidecognate of a first base type and a nucleotide cognate of a second basetype; (b) contacting the primed template nucleic acid with a polymeraseand a second mixture of nucleotides under ternary complex stabilizingconditions, wherein the second mixture includes a nucleotide cognate ofthe first base type and a nucleotide cognate of a third base type; (c)examining products of steps (a) and (b) for signals produced by aternary complex that includes the primed template nucleic acid, apolymerase and a next correct nucleotide, wherein signals acquired forthe product of step (a) are ambiguous for the first and second basetype, and wherein signals acquired for the product of step (b) areambiguous for the first and third base type; (d) disambiguating signalsacquired in step (c) to identify a base type that binds the next correctnucleotide; (e) adding a next correct nucleotide to the primer of theprimed template nucleic acid after step (c), thereby producing anextended primer; and (f) repeating steps (a) through (e) for the primedtemplate nucleic acid that comprises the extended primer.

Further still, a method of nucleic acid sequencing can include the stepsof (a) contacting a primed template nucleic acid with a first mixtureincluding a polymerase, a nucleotide cognate of a first base type in thetemplate and a nucleotide cognate of a second base type in the template,wherein the contact occurs in a binding reaction that (i) stabilizesternary complexes including the primed template nucleic acid, thepolymerase and a next correct nucleotide, and (ii) preventsincorporation of the next correct nucleotide into the primer; (b)examining the binding reaction to determine whether a ternary complexformed; (c) subjecting the primed template nucleic acid to a repetitionof steps (a) and (b), wherein the first mixture is replaced with asecond mixture, the second mixture including a polymerase, a nucleotidecognate of the first base type in the template and a nucleotide cognateof a third base type in the template; (d) identifying the next correctnucleotide for the primed template nucleic acid using the examination ofthe binding reaction, or the product thereof, wherein (i) the nextcorrect nucleotide is identified as a cognate of the first base type ifternary complex is detected in step (b) and detected in the repetitionof step (b), (ii) the next correct nucleotide is identified as a cognateof the second base type if ternary complex is detected in step (b) andundetected in the repetition of step (b), and (iii) the next correctnucleotide is identified as a cognate of the third base type if ternarycomplex is undetected in step (b) and detected in the repetition of step(b); (e) adding a next correct nucleotide to the primer of the primedtemplate nucleic acid after step (c), thereby producing an extendedprimer; and (f) repeating steps (a) through (e) for the primed templatenucleic acid that comprises the extended primer.

The next correct nucleotide that is added to the primer in a sequencingmethod can be reversibly terminated, so as to produce an extended,reversibly terminated primer. Adding a reversibly terminated nucleotideto the 3′ end of the primer provides a means to prevent more than onenucleotide from being added to the primer during the extension step andfurther prevents unwanted extension of the primer in a subsequentexamination step. Thus, each position in the template can be examinedsequentially. Furthermore, a stabilized ternary complex can be formed ateach position and examined to detect the next correct nucleotide for thetemplate that is hybridized to the extended, reversibly terminatedprimer. The method can be repeated in a step-wise fashion by thenremoving or modifying the reversible terminator moiety from theextended, reversibly terminated primer to produce an extendible primer.

Typically, a reversibly terminated nucleotide that is added to a primerin a method set forth herein does not have an exogenous label. This isbecause the extended primer need not be detected in a method set forthherein. However, if desired, one or more types of reversibly terminatednucleotides used in a method set forth herein can be detected, forexample, via exogenous labels attached to the nucleotides. Exemplaryreversible terminator moieties, methods for incorporating them intoprimers and methods for modifying the primers for further extension(often referred to as ‘deblocking’) are set forth in U.S. Pat. Nos.7,544,794; 7,956,171; 8,034,923; 8,071,755; 8,808,989; or 9,399,798.Further examples are set forth in Bentley et al., Nature 456:53-59(2008), WO 04/018497; U.S. Pat. No. 7,057,026; WO 91/06678; WO07/123744; U.S. Pat. No. 7,329,492; U.S. Pat. No. 7,211,414; U.S. Pat.No. 7,315,019; U.S. Pat. No. 7,405,281, and US 2008/0108082, each ofwhich is incorporated herein by reference.

Other techniques for facilitating repetition of steps in a sequencingmethod set forth herein include, for example, forming a stabilizedternary complex that can be modified to an extension competent form. Forexample, an extendible primer can be present in a stabilized ternarycomplex, but extension can be prevented by the composition of thereaction mixture. In this case, extension can be facilitated bycontacting the stabilized ternary complex with a ternary complexdestabilizing agent to allow incorporation of the nucleotide in theternary complex, removing a ternary complex stabilizing agent to allowincorporation of the nucleotide in the ternary complex, or removing thenucleotide and/or polymerase from the stabilized ternary complex andintroducing another polymerase and/or nucleotide under conditions thatfacilitate extension of the primer.

A primer extension step can be carried out by contacting a primedtemplate nucleic acid with an extension reaction mixture. In some cases,the fluid that was present in the examination step is removed andreplaced with the extension reaction mixture. Alternatively, theextension reaction mixture can be formed by adding one or more reagentsto the fluid that was present in the examination step. Optionally, theincorporation reaction mixture includes a different composition ofnucleotides than an examination step. For example, an examination stepcan include one or more nucleotide types that are not present in theincorporation reaction and vice versa. By way of more specific example,an examination step can omit at least one type of nucleotide and aprimer extension step can employ at least four types of nucleotides.Optionally, one or more nucleotide types is added to an examinationmixture for a primer extension step.

Nucleotides present in an examination step may cause unwanted nucleotideincorporation if carried over into an extension step. Thus, a wash stepcan be employed prior to a primer extension step to remove nucleotides.Optionally, free nucleotides may be removed by enzymes such asphosphatases, by chemical modification or by physical separationtechniques.

Optionally, a nucleotide enclosed within a stabilized ternary complex ofan examination step is incorporated into the 3′-end of a primer during asubsequent primer extension step. Alternatively, a primer extension stepincludes replacing a nucleotide from a prior examination step andincorporating another nucleotide (of the same or different type) intothe 3′-end of the primer.

Optionally, a polymerase present during an examination step is removedand replaced with a different polymerase for a subsequent primerextension step. Alternatively, the polymerase present during theexamination step is retained and modified for a subsequent incorporationstep. Optionally, one or more nucleotides present during an examinationstep are modified for a subsequent primer extension step. A fluid,reagent or condition that is present during an examination step may bealtered by any of a variety of techniques for use in a subsequent primerextension step. Exemplary techniques include, but are not limited to,removing reagents, chelating reagents, diluting reagents, addingreagents, altering reaction conditions such as temperature, ionicstrength, conductivity or pH, or any combination thereof. The reagentsin a reaction mixture including any combination of polymerase, primedtemplate nucleic acid, and nucleotide may be modified during anexamination step and/or primer extension step.

Typically, an extension step employed in a method set forth herein willresult in addition of a nucleotide cognate for any base type that isexpected to be present in a template nucleic acid. For example, primerextension can be carried out under conditions that result inincorporation of cognate nucleotides for all four base types that arepresent in DNA (e.g. adenine, thymine, guanine and cytosine) or RNA(e.g. adenine, uracil, guanine and cytosine). The different nucleotidetypes can be present simultaneously in an extension reaction, or theycan participate in serial extension reactions. For example, some or allof the nucleotide types can been delivered simultaneously in a singleextension reaction. Alternatively, different nucleotide types can beserially delivered (individually or in subsets) such that they arecombined into a single extension reaction or such that serial extensionreactions occur.

Although extension has been exemplified above with regard to the use ofcognates for four base types in a template, it will be understood that alarger repertoire of nucleotides can be used. The number of nucleotidetypes can increase, for example, when using templates having one or bothmembers of an unnatural base pair. In some embodiments, it may bedesirable to extend a primer with cognates for only a subset of basetypes that are expected to be present in a template. Thus, it ispossible to include cognates for fewer than 6, 5, 4, 3 or 2 base types.Alternatively or additionally, a method set forth herein can be used toextend a primer with cognates for at least 1, 2, 3, 4, 5, 6 or more basetypes.

A sequencing method can include multiple repetitions of steps set forthherein. For example, examination and extension steps can be repeatedmultiple times as can optional steps of deblocking primers, or washingaway unwanted reactants or products between various steps. Accordingly,a primed template nucleic acid can be subjected at least 2, 5, 10, 25,50, 100 or more steps of a method set forth herein. Not all of the stepsneed to be repeated nor do repeated steps need to occur in the sameorder in each repetition. For example, next correct nucleotides at eachposition of a template can be identified using real time analysis (i.e.in parallel with fluidic and detection steps of a sequencing method).However, real time analysis is not necessary and instead next correctnucleotides can be identified after some or all of the fluidic anddetection steps have been completed. Accordingly, signals from at leastsome sequencing by binding cycles can be disambiguated and/or theidentity of nucleotide types for at least some cycles can be imputedwhile fluidic steps are occurring. Optionally, signals can bedisambiguated and/or the identity of non-detected nucleotide types canbe imputed after some or all of the fluidic and detection cycles havebeen completed.

A primer extension step need not use a labeled polymerase. For example,a polymerase that is used for an extension step need not be attached toan exogenous label (e.g. covalently or otherwise). However, a polymerasethat is used for primer extension can include an exogenous label, forexample, a label that was used in a previous examination step.

As set forth above, different activities of polymerases can be exploitedin a method set forth herein. The different activities can follow fromdifferences in the structure (e.g. via natural activities, mutations orchemical modifications). Nevertheless, polymerase can be obtained from avariety of known sources and applied in accordance with the teachingsset forth herein and recognized activities of polymerases. Useful DNApolymerases include, but are not limited to, bacterial DNA polymerases,eukaryotic DNA polymerases, archaeal DNA polymerases, viral DNApolymerases and phage DNA polymerases. Bacterial DNA polymerases includeE. coli DNA polymerases I, II and III, IV and V, the Klenow fragment ofE. coli DNA polymerase, Clostridium stercorarium (Cst) DNA polymerase,Clostridium thermocellum (Cth) DNA polymerase and Sulfolobussolfataricus (Sso) DNA polymerase. Eukaryotic DNA polymerases includeDNA polymerases α, β, γ, δ, €, η, ζ, λ, σ, μ, and k, as well as the Revlpolymerase (terminal deoxycytidyl transferase) and terminaldeoxynucleotidyl transferase (TdT). Viral DNA polymerases include T4 DNApolymerase, phi-29 DNA polymerase, GA-1, phi-29-like DNA polymerases,PZA DNA polymerase, phi-15 DNA polymerase, Cpl DNA polymerase, Cp7 DNApolymerase, T7 DNA polymerase, and T4 polymerase. Other useful DNApolymerases include thermostable and/or thermophilic DNA polymerasessuch as Thermus aquaticus (Taq) DNA polymerase, Thermus filiformis (Tfi)DNA polymerase, Thermococcus zilligi (Tzi) DNA polymerase, Thermusthermophilus (Tth) DNA polymerase, Thermus flavusu (Tfl) DNA polymerase,Pyrococcus woesei (Pwo) DNA polymerase, Pyrococcus furiosus (Pfu) DNApolymerase and Turbo Pfu DNA polymerase, Thermococcus litoralis (Tli)DNA polymerase, Pyrococcus sp. GB-D polymerase, Thermotoga maritima(Tma) DNA polymerase, Bacillus stearothermophilus (Bst) DNA polymerase,Pyrococcus Kodakaraensis (KOD) DNA polymerase, Pfx DNA polymerase,Thermococcus sp. JDF-3 (JDF-3) DNA polymerase, Thermococcus gorgonarius(Tgo) DNA polymerase, Thermococcus acidophilium DNA polymerase;Sulfolobus acidocaldarius DNA polymerase; Thermococcus sp. go N-7 DNApolymerase; Pyrodictium occultum DNA polymerase; Methanococcus voltaeDNA polymerase; Methanococcus thermoautotrophicum DNA polymerase;Methanococcus jannaschii DNA polymerase; Desulfurococcus strain TOK DNApolymerase (D. Tok Pol); Pyrococcus abyssi DNA polymerase; Pyrococcushorikoshii DNA polymerase; Pyrococcus islandicum DNA polymerase;Thermococcus fumicolans DNA polymerase; Aeropyrum pernix DNA polymerase;and the heterodimeric DNA polymerase DP1/DP2. Engineered and modifiedpolymerases also are useful in connection with the disclosed techniques.For example, modified versions of the extremely thermophilic marinearchaea Thermococcus species 9° N (e.g., Therminator DNA polymerase fromNew England BioLabs Inc.; Ipswich, Mass.) can be used. Still otheruseful DNA polymerases, including the 3PDX polymerase are disclosed inU.S. Pat. No. 8,703,461, the disclosure of which is incorporated hereinby reference.

Useful RNA polymerases include, but are not limited to, viral RNApolymerases such as T7 RNA polymerase, T3 polymerase, SP6 polymerase,and Kll polymerase; Eukaryotic RNA polymerases such as RNA polymerase I,RNA polymerase II, RNA polymerase III, RNA polymerase IV, and RNApolymerase V; and Archaea RNA polymerase.

Another useful type of polymerase is a reverse transcriptase. Exemplaryreverse transcriptases include, but are not limited to, HIV-1 reversetranscriptase from human immunodeficiency virus type 1 (PDB 1HMV), HIV-2reverse transcriptase from human immunodeficiency virus type 2, M-MLVreverse transcriptase from the Moloney murine leukemia virus, AMVreverse transcriptase from the avian myeloblastosis virus, andTelomerase reverse transcriptase that maintains the telomeres ofeukaryotic chromosomes.

A polymerase having an intrinsic 3′-5′ proofreading exonuclease activitycan be useful for some embodiments. Polymerases that substantially lack3′-5′ proofreading exonuclease activity are also useful in someembodiments, for example, in most genotyping and sequencing embodiments.Absence of exonuclease activity can be a wild type characteristic or acharacteristic imparted by a variant or engineered polymerase structure.For example, exo minus Klenow fragment is a mutated version of Klenowfragment that lacks 3′-5′ proofreading exonuclease activity. Klenowfragment and its exo minus variant can be useful in a method orcomposition set forth herein.

Examples of reagents and conditions that can be used for apolymerase-based primer extension step include, for example, those setforth in commonly owned U.S. Pat. App. Pub. No. 2017/0022553 A1 or U.S.Pat. App. Ser. Nos. 62/447,319; 62/440,624 or 62/450,397, each of whichis incorporated herein by reference. Other useful reagent and conditionsfor polymerase-based primer extension are set forth in Bentley et al.,Nature 456:53-59 (2008), WO 04/018497; WO 91/06678; WO 07/123744; U.S.Pat. Nos. 7,057,026; 7,329,492; 7,211,414; 7,315,019 or 7,405,281, andUS Pat. App. Pub. No. 2008/0108082 A1, each of which is incorporatedherein by reference.

Optionally, the provided methods further include one or more wash steps.A wash step can occur before or after any other step in the method. Forexample, a method set forth herein can optionally include a step ofwashing a solid support after forming one or more stabilized ternarycomplexes. The wash can provide the advantage of removing contaminantssuch as components of a mixture from which one or more components of thestabilized ternary complex were derived. In particular embodiments, thewash step occurs under conditions that stabilize the ternary complex.For example, one or more of the stabilizing conditions or stabilizingagents set froth elsewhere herein can be employed during a wash step.Optionally, the wash solution includes nucleotide(s) of the same type asthe next correct nucleotide(s) used during formation of the stabilizedternary complex. Including the next correct nucleotide(s) at asufficient concentration can provide the advantage of stabilizingpreviously formed ternary complexes from unwanted disassociation. Thisin turn prevents unwanted reduction in detection sensitivity due towashing away previously formed ternary complexes. Optionally, theternary complex has a half-life and the wash step is performed for aduration shorter than the half-life of the ternary complex. Wash stepscan also be carried out after examination or primer extension steps.

A stabilized ternary complex, or a component that is capable of forming(i.e. participating in the formation of) a ternary complex, can beattached to a solid support. The solid support can be made from any of avariety of materials set forth herein. Suitable materials may includeglass, polymeric materials, silicon, quartz (fused silica), borofloatglass, silica, silica-based materials, carbon, metals, an optical fiberor bundle of optical fibers, sapphire, or plastic materials. Theparticular material can be selected based on properties desired for aparticular use. For example, materials that are transparent to a desiredwavelength of radiation are useful for analytical techniques that willutilize radiation of that wavelength. Conversely, it may be desirable toselect a material that does not pass radiation of a certain wavelength(e.g. being opaque, absorptive or reflective). Other properties of amaterial that can be exploited are inertness or reactivity to certainreagents used in a downstream process, such as those set forth herein,or ease of manipulation, or low cost of manufacture.

A particularly useful solid support is a particle such as a bead ormicrosphere. Populations of beads can be used for attachment ofpopulations of stabilized ternary complexes or components capable offorming the complexes (e.g. polymerases, templates, primers ornucleotides). In some embodiments, it may be useful to use aconfiguration whereby each bead has a single type of stabilized ternarycomplex or a single type of component capable of forming the complex.For example, an individual bead can be attached to a single type ofternary complex, a single type of template allele, a single type ofallele-specific primer, a single type of locus-specific primer or asingle type of nucleotide. Alternatively, different types of componentsneed not be separated on a bead-by-bead basis. As such, a single beadcan bear multiple different types of ternary complexes, template nucleicacids, primers, primed template nucleic acids and/or nucleotides. Thecomposition of a bead can vary, depending for example, on the format,chemistry and/or method of attachment to be used. Exemplary beadcompositions include solid supports, and chemical functionalitiesimparted thereto, used in protein and nucleic acid capture methods. Suchcompositions include, for example, plastics, ceramics, glass,polystyrene, melamine, methylstyrene, acrylic polymers, paramagneticmaterials, thoria sol, carbon graphite, titanium dioxide, latex orcross-linked dextrans such as Sepharos™, cellulose, nylon, cross-linkedmicelles and Teflon™, as well as other materials set forth in“Microsphere Detection Guide” from Bangs Laboratories, Fishers Ind.,which is incorporated herein by reference.

The geometry of a particle, bead or microsphere also can correspond to awide variety of different forms and shapes. For example, they can besymmetrically shaped (e.g. spherical or cylindrical) or irregularlyshaped (e.g. controlled pore glass). In addition, beads can be porous,thus increasing the surface area available for capture of ternarycomplexes or components thereof. Exemplary sizes for beads used hereincan range from nanometers to millimeters or from about 10 nm-1 mm.

In particular embodiments, beads can be arrayed or otherwise spatiallydistinguished. Exemplary bead-based arrays that can be used include,without limitation, a BeadChip™ Array available from Illumina, Inc. (SanDiego, Calif.) or arrays such as those described in U.S. Pat. Nos.6,266,459; 6,355,431; 6,770,441; 6,859,570; or 7,622,294; or PCTPublication No. WO 00/63437, each of which is incorporated herein byreference. Beads can be located at discrete locations, such as wells, ona solid-phase support, whereby each location accommodates a single bead.Alternatively, discrete locations where beads reside can each include aplurality of beads as described, for example, in U.S. Pat. App. Pub.Nos. 2004/0263923 A1, 2004/0233485 A1, 2004/0132205 A1, or 2004/0125424A1, each of which is incorporated herein by reference.

As will be recognized from the above bead array embodiments, a method ofthe present disclosure can be carried out in a multiplex format wherebymultiple different types of nucleic acids are detected in parallel in amethod set forth herein. Although it is also possible to seriallyprocess different types of nucleic acids using one or more steps of themethods set forth herein, parallel processing can provide cost savings,time savings and uniformity of conditions. An apparatus or method of thepresent disclosure can include at least 2, 10, 100, 1×10³, 1×10⁴, 1×10⁵,1×10⁶, 1×10⁹, or more different nucleic acids. Alternatively oradditionally, an apparatus or method of the present disclosure caninclude at most 1×10⁹, 1×10⁶, 1×10⁵, 1×10⁴, 1×10³, 100, 10, 2 or fewer,different nucleic acids. Accordingly, various reagents or products setforth herein as being useful in the apparatus or methods (e.g. primedtemplate nucleic acids or stabilized ternary complexes) can bemultiplexed to have different types or species in these ranges.

Further examples of commercially available arrays that can be usedinclude, for example, an Affymetrix GeneChip™ array. A spotted array canalso be used according to some embodiments. An exemplary spotted arrayis a CodeLink™ Array available from Amersham Biosciences. Another arraythat is useful is one that is manufactured using inkjet printing methodssuch as SurePrint™ Technology available from Agilent Technologies.

Other useful arrays include those that are used in nucleic acidsequencing applications. For example, arrays that are used to attachamplicons of genomic fragments (often referred to as clusters) can beparticularly useful. Examples of nucleic acid sequencing arrays that canbe used herein include those described in Bentley et al., Nature456:53-59 (2008), PCT Pub. Nos. WO 91/06678; WO 04/018497 or WO07/123744; U.S. Pat. Nos. 7,057,026; 7,211,414; 7,315,019; 7,329,492 or7,405,281; or U.S. Pat. App. Pub. No. 2008/0108082, each of which isincorporated herein by reference.

A nucleic acid can be attached to a support in a way that providesdetection at a single molecule level or at an ensemble level. Forexample, a plurality of different nucleic acids can be attached to asolid support in a way that an individual stabilized ternary complexthat forms on one nucleic acid molecule on the support can bedistinguished from all neighboring ternary complexes that form on thenucleic acid molecules of the support. As such, one or more differenttemplates can be attached to a solid support in a format where eachsingle molecule template is physically isolated and detected in a waythat the single molecule is resolved from all other molecules on thesolid support.

Alternatively, a method of the present disclosure can be carried out forone or more nucleic acid ensembles, an ensemble being a population ofnucleic acids having a common template sequence. Cluster methods can beused to attach one or more ensembles to a solid support. As such, anarray can have a plurality of ensembles, each of the ensembles beingreferred to as a cluster or array feature in that format. Clusters canbe formed using methods known in the art such as bridge amplification oremulsion PCR. Useful bridge amplification methods are described, forexample, in U.S. Pat. Nos. 5,641,658 or 7,115,400; or U.S. Patent Pub.Nos. 2002/0055100 A1; 2004/0002090 A1; 2004/0096853 A1; 2007/0128624 A1;or 2008/0009420 A1. Emulsion PCR methods include, for example, methodsdescribed in Dressman et al., Proc. Natl. Acad. Sci. USA 100:8817-8822(2003), WO 05/010145, or U.S. Patent Pub. Nos. 2005/0130173 A1 or2005/0064460 A1, each of which is incorporated herein by reference inits entirety. Another useful method for amplifying nucleic acids on asurface is rolling circle amplification (RCA), for example, as describedin Lizardi et al., Nat. Genet. 19:225-232 (1998) or US 2007/0099208 A1,each of which is incorporated herein by reference.

In particular embodiments, a stabilized ternary complex, polymerase,nucleic acid or nucleotide is attached to a flow cell surface or to asolid support in a flow cell. A flow cell allows convenient fluidicmanipulation by passing solutions into and out of a fluidic chamber thatcontacts the support-bound, ternary complex. The flow cell also providesfor detection of the fluidically manipulated components. For example, adetector can be positioned to detect signals from the solid support,such as signals from a label that is recruited to the solid support dueto formation of a stabilized ternary complex. Exemplary flow cells thatcan be used are described, for example, in US Pat. App. Pub. No.2010/0111768 A1, WO 05/065814 or US Pat. App. Pub. No. 2012/0270305 A1,each of which is incorporated herein by reference.

Nucleic acids that are used in a method or composition herein can be DNAsuch as genomic DNA, synthetic DNA, amplified DNA, copy DNA (cDNA) orthe like. RNA can also be used such as mRNA, ribosomal RNA, tRNA or thelike. Nucleic acid analogs can also be used as templates herein. Thus,template nucleic acids used herein can be derived from a biologicalsource, synthetic source or amplification product. Primers used hereincan be DNA, RNA or analogs thereof.

Particularly useful nucleic acid templates are genome fragments thatinclude sequences identical to a portion of a genome. A population ofgenome fragments can include at least 5%, 10%, 20%, 30%, or 40%, 50%,60%, 70%, 75%, 80%, 85%, 90%, 95% or 99% of a genome. A genome fragmentcan have, for example, a sequence that is substantially identical to atleast about 25, 50, 70, 100, 200, 300, 400, 500, 600, 700, 800, 900 or1000 or more nucleotides of a genome. A genome fragment can be DNA, RNA,or an analog thereof.

Exemplary organisms from which nucleic acids can be derived include, forexample, those from a mammal such as a rodent, mouse, rat, rabbit,guinea pig, ungulate, horse, sheep, pig, goat, cow, cat, dog, primate,human or non-human primate; a plant such as Arabidopsis thaliana, corn,sorghum, oat, wheat, rice, canola, or soybean; an algae such asChlamydomonas reinhardtii; a nematode such as Caenorhabditis elegans; aninsect such as Drosophila melanogaster, mosquito, fruit fly, honey beeor spider; a fish such as zebrafish; a reptile; an amphibian such as afrog or Xenopus laevis; a dictyostelium discoideum; a fungi such aspneumocystis carinii, Takifugu rubripes, yeast, Saccharamoycescerevisiae or Schizosaccharomyces pombe; or a plasmodium falciparum.Nucleic acids can also be derived from a prokaryote such as a bacterium,Escherichia coli, staphylococci or mycoplasma pneumoniae; an archae; avirus such as Hepatitis C virus or human immunodeficiency virus; or aviroid. Nucleic acids can be derived from a homogeneous culture orpopulation of the above organisms or alternatively from a collection ofseveral different organisms, for example, in a community or ecosystem.Nucleic acids can be isolated using methods known in the art including,for example, those described in Sambrook et al., Molecular Cloning: ALaboratory Manual, 3rd edition, Cold Spring Harbor Laboratory, New York(2001) or in Ausubel et al., Current Protocols in Molecular Biology,John Wiley and Sons, Baltimore, Md. (1998), each of which isincorporated herein by reference.

A template nucleic acid can be obtained from a preparative method suchas genome isolation, genome fragmentation, gene cloning and/oramplification. The template can be obtained from an amplificationtechnique such as polymerase chain reaction (PCR), rolling circleamplification (RCA), multiple displacement amplification (MDA) or thelike. Exemplary methods for isolating, amplifying and fragmentingnucleic acids to produce templates for analysis on an array are setforth in U.S. Pat. Nos. 6,355,431 or 9,045,796, each of which isincorporated herein by reference. Amplification can also be carried outusing a method set forth in Sambrook et al., Molecular Cloning: ALaboratory Manual, 3rd edition, Cold Spring Harbor Laboratory, New York(2001) or in Ausubel et al., Current Protocols in Molecular Biology,John Wiley and Sons, Baltimore, Md. (1998), each of which isincorporated herein by reference.

The present disclosure provides systems for detecting nucleic acids, forexample, using methods set forth herein. For example, a system can beconfigured for genotyping reactions or sequencing by binding reactionsinvolving the examination of the interaction between a polymerase and aprimed template nucleic acid in the presence of nucleotides to identifythe next base in the template nucleic acid sequence. Optionally, asystem includes components and reagents for performing one or more stepsset forth herein including, but not limited to, forming at least onestabilized ternary complex between a primed template nucleic acid,polymerase and next correct nucleotide, detecting the stabilized ternarycomplex(es), extending the primer of each primed template with a nextcorrect nucleotide, and/or identifying a nucleotide or sequence ofnucleotides present in the template.

A system of the present disclosure can include a vessel or solid supportfor carrying out a nucleic acid detection method. For example, thesystem can include an array, flow cell, multi-well plate or otherconvenient apparatus. The vessel or solid support can be removable,thereby allowing it to be placed into or removed from the system. Assuch, a system can be configured to sequentially process a plurality ofvessels or solid supports. The system can include a fluidic systemhaving reservoirs for containing one or more of the reagents set forthherein (e.g. polymerase, primer, template nucleic acid, nucleotide(s)for ternary complex formation, nucleotides for primer extension,deblocking reagents or mixtures of such components). The fluidic systemcan be configured to deliver reagents to a vessel or solid support, forexample, via channels or droplet transfer apparatus (e.g. electrowettingapparatus). Any of a variety of detection apparatus can be configured todetect the vessel or solid support where reagents interact. Examplesinclude luminescence detectors, surface plasmon resonance detectors andothers known in the art. Exemplary systems having fluidic and detectioncomponents that can be readily modified for use in a system hereininclude, but are not limited to, those set forth in U.S. Pat. App. Ser.No. 62/481,289; U.S. Pat. Nos. 8,241,573; 7,329,860 or 8,039,817; or USPat. App. Pub. Nos. 2009/0272914 A1 or 2012/0270305 A1, each of which isincorporated herein by reference.

Optionally, a system of the present disclosure further includes acomputer processing unit (CPU) that is configured to operate systemcomponents. The same or different CPU can interact with the system toacquire, store and process signals (e.g. signals detected in a methodset forth herein). In particular embodiments, a CPU can be used todetermine, from the signals, the identify the nucleotide that is presentat a particular location in a template nucleic acid. In some cases, theCPU will identify a sequence of nucleotides for the template from thesignals that are detected. In particular embodiments, the CPU isprogrammed to compare signals obtained from different binding reactionsto disambiguate signals, thereby identifying nucleotides at one or moreposition in a template nucleic acid. Alternatively or additionally, aCPU can be programmed to compare signals obtained from different bindingreactions to identify a nucleotide at one or more position in a templatenucleic acid by imputation.

A useful CPU can include one or more of a personal computer system,server computer system, thin client, thick client, hand-held or laptopdevice, multiprocessor system, microprocessor-based system, set top box,programmable consumer electronic, network PC, minicomputer system,mainframe computer system, smart phone, and distributed cloud computingenvironments that include any of the above systems or devices, and thelike. The CPU can include one or more processors or processing units, amemory architecture that may include RAM and non-volatile memory. Thememory architecture may further include removable/non-removable,volatile/non-volatile computer system storage media. Further, the memoryarchitecture may include one or more readers for reading from andwriting to a non-removable, non-volatile magnetic media, such as a harddrive, a magnetic disk drive for reading from and writing to aremovable, non-volatile magnetic disk, and/or an optical disk drive forreading from or writing to a removable, non-volatile optical disk suchas a CD-ROM or DVD-ROM. The CPU may also include a variety of computersystem readable media. Such media may be any available media that isaccessible by a cloud computing environment, such as volatile andnon-volatile media, and removable and non-removable media.

The memory architecture may include at least one program product havingat least one program module implemented as executable instructions thatare configured to carry out one or more steps of a method set forthherein. For example, executable instructions may include an operatingsystem, one or more application programs, other program modules, andprogram data. Generally, program modules may include routines, programs,objects, components, logic, data structures, and so on, that performparticular tasks such as processing of signals detected in a method setforth herein, disambiguating signals to identify nucleotides or imputingnucleotide identity where signals for other types of nucleotides aredetected.

The components of a CPU may be coupled by an internal bus that may beimplemented as one or more of any of several types of bus structures,including a memory bus or memory controller, a peripheral bus, anaccelerated graphics port, and a processor or local bus using any of avariety of bus architectures. By way of example, and not limitation,such architectures include Industry Standard Architecture (ISA) bus,Micro Channel Architecture (MCA) bus, Enhanced ISA (EISA) bus, VideoElectronics Standards Association (VESA) local bus, and PeripheralComponent Interconnects (PCI) bus.

A CPU can optionally communicate with one or more external devices suchas a keyboard, a pointing device (e.g. a mouse), a display, such as agraphical user interface (GUI), or other device that facilitatesinteraction of a use with the nucleic acid detection system. Similarly,the CPU can communicate with other devices (e.g., via network card,modem, etc.). Such communication can occur via I/O interfaces. Stillyet, a CPU of a system herein may communicate with one or more networkssuch as a local area network (LAN), a general wide area network (WAN),and/or a public network (e.g., the Internet) via a suitable networkadapter.

This disclosure further provides a kit for distinguishing nucleotides ina nucleic acid template. The kit can include reagents for carrying outone or more of the methods set forth herein. For example, a kit caninclude reagents for producing a stabilized ternary complex when mixedwith one or more primed template nucleic acid. More specifically, a kitcan include one or more of the mixtures of nucleotides used in a methodset forth herein, including for example, the methods set forth in theExamples section below. In addition to the nucleotide mixtures the kitcan include a polymerase that is capable of forming a stabilized ternarycomplex. The nucleotides, polymerase or both can include an exogenouslabel, for example, as set forth herein in the context of variousmethods.

In some embodiments, the kit can be configured to support a repetitivemethod such as a sequencing by binding method. Accordingly, a kit canfurther include reagents for carrying out primer extension. Exemplaryreagents for primer extension can include a polymerase and mixture offour nucleotide types. The nucleotide types used for extension canoptionally include reversible terminating groups. In this option, thekit can further include reagents for deblocking a primer that hasincorporated the reversibly terminated nucleotides.

Accordingly, any of the components or articles used in performing themethods set forth herein can be usefully packaged into a kit. Forexample, the kits can be packed to include some, many or all of thecomponents or articles used in performing the methods set forth herein.Exemplary components include, for example, nucleotides, polymerases,terminator moieties, deblocking reagents and the like as set forthherein and in references cited herein. Any of such reagents can include,for example, some, many or all of the buffers, components and/orarticles used for performing one or more of the subsequent steps foranalysis of a primed template nucleic acid. A kit need not include aprimer or template nucleic acid. Rather, a user of the kit can provide aprimed template nucleic acid which is to be combined with components ofthe kit.

One or more ancillary reagents also can be included in a kit. Suchancillary reagents can include any of the reagents exemplified aboveand/or other types of reagents useful in performing the methods setforth herein. Instructions can further be included in a kit. Theinstructions can include, for example, procedures for making anycomponents or articles used in the methods set forth herein, performingone or more steps of any embodiment of the methods set forth hereinand/or instructions for performing any of the subsequent analysis stepsemploying a primed template nucleic acid.

In particular embodiments, a kit includes a cartridge having reservoirsto contain the reagents and further having fluidic components fortransferring reagents from the reservoirs to a detection instrument. Forexample, the fluidic components can be configured to transfer reagentsto a flow cell where stabilized ternary complexes are detected. Anexemplary fluidic cartridge that can be included in a kit (or system) ofthe present disclosure is described in U.S. Pat. App. Ser. No.62/481,289, which is incorporated herein by reference.

NUMBERED EMBODIMENTS Embodiment 1

A method of nucleic acid detection, comprising steps of:

(a) contacting a primed template nucleic acid with a polymerase and afirst mixture of nucleotides under conditions for stabilizing a ternarycomplex at a nucleotide position in the template, wherein the firstmixture comprises a nucleotide cognate of a first base type and anucleotide cognate of a second base type;

(b) contacting the primed template nucleic acid with a polymerase and asecond mixture of nucleotides under conditions for stabilizing a ternarycomplex at the nucleotide position in the template, wherein the secondmixture comprises a nucleotide cognate of the first base type and anucleotide cognate of a third base type;

(c) examining products of steps (a) and (b) for signals produced by aternary complex that comprises the primed template nucleic acid, apolymerase and a next correct nucleotide, wherein signals acquired forthe product of step (a) are ambiguous for the first and second basetype, and wherein signals acquired for the product of step (b) areambiguous for the first and third base type;

(d) disambiguating signals acquired in step (c) to identify a base typethat binds the next correct nucleotide.

Embodiment 2

The method of embodiment 1, wherein the primed template nucleic acid isnot in contact with a nucleotide cognate of a fourth base type duringstep (c).

Embodiment 3

The method of embodiment 2, wherein (i) the first base type iscorrelated with presence of signals for the product of step (a) andpresence of signals for the product of step (b),

(ii) the second base type is correlated with presence of signals for theproduct of step (a) and absence of signals for the product of step (b),and

(iii) the third base type is correlated with absence of signals for theproduct of step (a) and presence of signals for the product of step (b).

Embodiment 4

The method of embodiment 3, wherein the first mixture lacks nucleotidecognates of the third and fourth base types, and wherein the secondmixture lacks nucleotide cognates of the second and fourth base types.

Embodiment 5

The method of embodiment 4, wherein (iv) the fourth base type iscorrelated with absence of signals for the product of step (a) andabsence of signals for the product of step (b).

Embodiment 6

The method of embodiment 1, wherein the first mixture further comprisesnucleotide cognates of the third and fourth base types,

wherein the product of step (a) produces a first signal for stabilizedternary complex that comprises the nucleotide cognate of the first basetype and for stabilized ternary complex that comprises the nucleotidecognate of the second base type,

wherein the product of step (a) produces a second signal for stabilizedternary complex that comprises the nucleotide cognate of the third basetype and for stabilized ternary complex that comprises the nucleotidecognate of the fourth base type, and

wherein the examining of the products of step (a) distinguishes thefirst signal from the second signal.

Embodiment 7

The method of embodiment 6, wherein the second mixture further comprisesnucleotide cognates of the second and fourth base types,

wherein the product of step (b) produces the first signal for stabilizedternary complex that comprises the nucleotide cognate of the first basetype and for stabilized ternary complex that comprises the nucleotidecognate of the third base type, and

wherein the product of step (b) produces the second signal forstabilized ternary complex that comprises the nucleotide cognate of thesecond base type and for stabilized ternary complex that comprises thenucleotide cognate of the fourth base type.

Embodiment 8

The method of embodiment 7, wherein (i) the first base type iscorrelated with presence of the first signal for the products of steps(a) and (b),

(ii) the second base type is correlated with presence of the firstsignal for the product of step (a) and presence of the second signal forthe product of step (b),

(iii) the third base type is correlated with presence of the secondsignal for the product of step (a) and presence of the first signal forthe product of step (b), and

(iv) the fourth base type is correlated with presence of the secondsignal for the product of steps (a) and (b).

Embodiment 9

The method of embodiment 6, wherein the second mixture further comprisesnucleotide cognates of the second and fourth base types,

wherein the product of step (b) produces the first signal for stabilizedternary complex that comprises the nucleotide cognate of the first basetype and for stabilized ternary complex that comprises the nucleotidecognate of the third base type, and

wherein the product of step (b) produces the no signal for stabilizedternary complex that comprises the nucleotide cognate of the second basetype and for stabilized ternary complex that comprises the nucleotidecognate of the fourth base type.

Embodiment 10

The method of embodiment 9, wherein (i) the first base type iscorrelated with presence of the first signal for the products of steps(a) and (b),

(ii) the second base type is correlated with presence of the firstsignal for the product of step (a) and absence of signal for the productof step (b),

(iii) the third base type is correlated with presence of the secondsignal for the product of step (a) and presence of the first signal forthe product of step (b), and

(iv) the fourth base type is correlated with presence of the secondsignal for the product of step (a) and absence of signal for the productof step (b).

Embodiment 11

The method of any one of embodiments 1 to 10, wherein the signalsacquired in step (c) are produced by exogenous labels attached topolymerases.

Embodiment 12

The method of embodiment 1 or 11, wherein the signals for the productsof step (a) are acquired by a detector that is also used to detect thesignals for the products of step (b).

Embodiment 13

The method of any one of embodiments 1 to 12, wherein the signals forthe products of steps (a) and (b) comprise luminescent signals.

Embodiment 14

The method of any one of embodiments 1 to 13, wherein the nucleotides inthe first mixture do not comprise exogenous labels.

Embodiment 15

The method of embodiment 14, wherein the nucleotides in the secondmixture do not comprise exogenous labels.

Embodiment 16

The method of any one of embodiments 1 to 13, wherein the first mixturedoes not comprise labels that distinguish the nucleotide cognate of thefirst base type from the nucleotide cognate of the second base type.

Embodiment 17

The method of embodiment 16, wherein the second mixture does notcomprise labels that distinguish the nucleotide cognate of the firstbase type from the nucleotide cognate of the third base type.

Embodiment 18

The method of any one of embodiments 1 to 17, further comprising (e)adding a reversibly terminated, next correct nucleotide to the primer ofthe primed template nucleic acid after step (c), thereby producing anextended, reversibly terminated primer.

Embodiment 19

The method of embodiment 18, further comprising repeating steps (a)through (c) for the primed template nucleic acid that comprises theextended, reversibly terminated primer.

Embodiment 20

The method of embodiment 19, further comprising (f) removing thereversible terminator moiety from the extended, reversibly terminatedprimer after steps (a) through (c) are repeated.

Embodiment 21

The method of embodiment 19, wherein step (e) is carried out prior tostep (d).

Embodiment 22

The method of any one of embodiments 1 to 21, wherein the polymerase ofstep (a) is replaced with the polymerase of step (b).

Embodiment 23

The method of any one of embodiments 1 to 22, wherein the same type ofpolymerase is present in steps (a) and (b).

Embodiment 24

The method of embodiment 1, comprising a further step of contacting theprimed template nucleic acid with a polymerase and a nucleotide cognateof a fourth base type, wherein step (c) further comprises examiningproducts of the further step for signals produced by a ternary complexthat comprises the primed template nucleic acid, a polymerase and a nextcorrect nucleotide.

Embodiment 25

The method of embodiment 24, wherein (iv) the fourth base type iscorrelated with presence of signals for the product of the further step.

Embodiment 26

The method of any one of embodiments 1 to 25, wherein the steps arecarried out for a plurality of primed template nucleic acids havingdifferent sequences.

Embodiment 27

The method of embodiment 26, wherein the plurality of primed templatenucleic acids is attached to an array.

Embodiment 28

The method of any one of embodiments 1 to 27, further comprisingremoving the first mixture from the primed template nucleic acid priorto step (b).

Embodiment 29

The method of any one of embodiments 1 to 28, wherein the examining ofthe products of step (a) is carried out prior to step (b).

Embodiment 30

The method of embodiment 1, further comprising

(i) contacting the primed template nucleic acid with a polymerase and athird mixture of nucleotides under conditions for stabilizing a ternarycomplex at the nucleotide position in the template, wherein the thirdmixture comprises a nucleotide cognate of the second base type and anucleotide cognate of a fourth base type;

(ii) contacting the primed template nucleic acid with a polymerase and afourth mixture of nucleotides under conditions for stabilizing a ternarycomplex at the nucleotide position in the template, wherein the fourthmixture comprises a nucleotide cognate of the third base type and anucleotide cognate of the fourth base type; and

(iii) examining products of steps (i) and (ii) for signals produced by aternary complex that comprises the primed template nucleic acid, apolymerase and a next correct nucleotide, wherein signals acquired forthe product of step (i) are ambiguous for the second and fourth basetype, and wherein signals acquired for the product of step (ii) areambiguous for the third and fourth base type.

Embodiment 31

The method of embodiment 30, wherein the first mixture lacks nucleotidecognates of the third or fourth base types.

Embodiment 32

The method of embodiment 31, wherein the second mixture lacks nucleotidecognates of the second or fourth base types, wherein the third mixturelacks nucleotide cognates of the first or third base types, and whereinthe fourth mixture lacks nucleotide cognates of the first or second basetypes.

Embodiment 33

The method of embodiment 30, wherein the first mixture comprises alabeled nucleotide cognate of the first base type and a labelednucleotide cognate of the second base type, and wherein the firstmixture comprises a non-labeled nucleotide cognate of the third orfourth base types.

Embodiment 34

The method of embodiment 33, wherein the second mixture comprises alabeled nucleotide cognate of the first base type and a labelednucleotide cognate of the third base type, wherein the second mixturecomprises a non-labeled nucleotide cognate of the second or fourth basetypes,

Embodiment 35

The method of embodiment 34, wherein the third mixture comprises alabeled nucleotide cognate of the second base type and a labelednucleotide cognate of the fourth base type, wherein the third mixturecomprises a non-labeled nucleotide cognate of the first or third basetypes.

Embodiment 36

The method of embodiment 35, wherein the fourth mixture comprises alabeled nucleotide cognate of the third base type and a labelednucleotide cognate of the fourth base type, wherein the fourth mixturecomprises a non-labeled nucleotide cognate of the first or second basetypes.

Embodiment 37

A method of nucleic acid detection, comprising:

(a) forming a mixture under ternary complex stabilizing conditions,wherein the mixture comprises a primed template nucleic acid, apolymerase and nucleotide cognates of first, second and third base typesin the template;

(b) examining the mixture to determine whether a ternary complex formed;and

(c) identifying the next correct nucleotide for the primed templatenucleic acid molecule, wherein the next correct nucleotide is identifiedas a cognate of the first, second or third base type if ternary complexis detected in step (b), and wherein the next correct nucleotide isimputed to be a nucleotide cognate of a fourth base type based on theabsence of a ternary complex in step (b).

Embodiment 38

The method of embodiment 37, wherein the primed template nucleic acid isnot in contact with a nucleotide cognate of the fourth base type duringstep (b).

Embodiment 39

The method of embodiment 37 or 38, wherein the polymerase is attached toan exogenous label.

Embodiment 40

The method of embodiment 39, wherein the nucleotide cognates do notcomprise exogenous labels.

Embodiment 41

The method of embodiment 37 or 38, wherein the nucleotide cognatescomprise exogenous labels that distinguish the cognates of first, secondand third base types from each other.

Embodiment 42

The method of any one of embodiments 37 to 41, further comprising (d)adding a reversibly terminated, next correct nucleotide to the primer ofthe primed template nucleic acid after step (b), thereby producing anextended, reversibly terminated primer.

Embodiment 43

The method of embodiment 42, further comprising repeating steps (a) and(b) for the primed template nucleic acid that comprises the extended,reversibly terminated primer.

Embodiment 44

The method of embodiment 43, further comprising (e) removing thereversible terminator moiety from the extended, reversibly terminatedprimer after steps (a) and (b) are repeated.

Embodiment 45

The method of embodiment 42, wherein step (d) is carried out prior tostep (c).

Embodiment 46

The method of any one of embodiments 37 to 45, wherein the steps arecarried out for a plurality of primed template nucleic acids havingdifferent sequences.

Embodiment 47

The method of embodiment 46, wherein the plurality of primed templatenucleic acids is attached to an array.

Embodiment 48

A method of nucleic acid detection, comprising:

(a) sequentially contacting a primed template nucleic acid with at leasttwo separate mixtures under ternary complex stabilizing conditions,wherein the at least two separate mixtures each comprise a polymeraseand a nucleotide, whereby the sequentially contacting results in theprimed template nucleic acid being contacted, under the ternary complexstabilizing conditions, with nucleotide cognates for first, second andthird base types in the template;

(b) examining the at least two separate mixtures to determine whether aternary complex formed; and

(c) identifying the next correct nucleotide for the primed templatenucleic acid molecule, wherein the next correct nucleotide is identifiedas a cognate of the first, second or third base type if ternary complexis detected in step (b), and wherein the next correct nucleotide isimputed to be a nucleotide cognate of a fourth base type based on theabsence of a ternary complex in step (b).

Embodiment 49

The method of embodiment 48, wherein the primed template nucleic acid isnot in contact with a nucleotide cognate of the fourth base type duringstep (b).

Embodiment 50

The method of embodiment 48 or 49, wherein the polymerase is attached toan exogenous label.

Embodiment 51

The method of embodiment 50, wherein the nucleotide cognates do notcomprise exogenous labels.

Embodiment 52

The method of any one of embodiment 48 to 50, wherein the nucleotidecognates comprise exogenous labels that distinguish the cognates offirst, second and third base types from each other.

Embodiment 53

The method of any one of embodiments 48 to 52, further comprising (d)adding a reversibly terminated, next correct nucleotide to the primer ofthe primed template nucleic acid after step (b), thereby producing anextended, reversibly terminated primer.

Embodiment 54

The method of embodiment 53, further comprising repeating steps (a) and(b) for the primed template nucleic acid that comprises the extended,reversibly terminated primer.

Embodiment 55

The method of embodiment 54, further comprising (e) removing thereversible terminator moiety from the extended, reversibly terminatedprimer after steps (a) and (b) are repeated.

Embodiment 56

The method of embodiment 53, wherein step (d) is carried out prior tostep (c).

Embodiment 57

The method of any one of embodiments 48 to 56, wherein the steps arecarried out for a plurality of primed template nucleic acids havingdifferent sequences.

Embodiment 58

The method of embodiment 57, wherein the plurality of primed templatenucleic acids is attached to an array.

Embodiment 59

The method of embodiment 48, wherein the sequentially contacting of theprimed template nucleic acid with the at least two separate mixturescomprises:

(i) contacting the primed template nucleic acid with a polymerase and afirst mixture of nucleotides under ternary complex stabilizingconditions, wherein the first mixture comprises a nucleotide cognate ofa first base type and a nucleotide cognate of a second base type, and

(ii) contacting the primed template nucleic acid with a polymerase and asecond mixture of nucleotides under ternary complex stabilizingconditions, wherein the second mixture comprises a nucleotide cognate ofthe first base type and a nucleotide cognate of a third base type.

Embodiment 60

The method of embodiment 59, wherein step (b) comprises detectingsignals from the ternary complexes, wherein the signals do notdistinguish ternary complexes comprising the nucleotide cognate of thefirst base type from ternary complexes comprising the nucleotide cognateof the second base type.

Embodiment 61

The method of embodiment 60, wherein the signals do not distinguishternary complexes comprising the nucleotide cognate of the first basetype from ternary complexes comprising the nucleotide cognate of thethird base type.

Embodiment 62

The method of embodiment 59, wherein the nucleotides in the firstmixture do not comprise exogenous labels.

Embodiment 63

The method of embodiment 62, wherein the nucleotides in the secondmixture do not comprise exogenous labels.

Embodiment 64

The method of embodiment 59, wherein the first mixture does not compriselabels that distinguish the nucleotide cognate of the first base typefrom the nucleotide cognate of the second base type.

Embodiment 65

The method of embodiment 64, wherein the second mixture does notcomprise labels that distinguish the nucleotide cognate of the firstbase type from the nucleotide cognate of the third base type.

Embodiment 66

The method of embodiment 59, wherein the examination comprises detectingsignals from a label attached to the nucleotide cognate of the firstbase type that are the same as signals detected for a label attached tothe nucleotide cognate of the second base type.

Embodiment 67

The method of embodiment 66, wherein the examination comprises detectingsignals from a label attached to the nucleotide cognate of the firstbase type that are the same as signals detected for a label attached tothe nucleotide cognate of the third base type.

Embodiment 68

The method of embodiment 59, wherein the same type of polymerase is usedin (i) and in (ii).

Embodiment 69

The method of embodiment 59, wherein the type of polymerase in (i) isdifferent from the type of polymerase in (ii).

Embodiment 70

The method of embodiment 59, wherein the signals for the products of (i)are acquired by a detector that is also used to detect the signals forthe products of (ii).

Embodiment 71

A method of nucleic acid detection, comprising:

(a) sequentially contacting a primed template nucleic acid with firstand second mixtures under ternary complex stabilizing conditions,wherein each of the mixtures comprises a polymerase and nucleotidecognates for at least two of four different base types in the primedtemplate nucleic acid, wherein the mixtures differ by at least one typeof nucleotide cognate;

(b) examining the first and second mixtures, or products thereof,separately to detect ternary complexes; and

(c) identifying the next correct nucleotide for the primed templatenucleic acid molecule, wherein the next correct nucleotide is identifiedas a cognate of one of the four different base types if ternary complexis detected in the two mixtures.

Embodiment 72

The method of embodiment 71, wherein the first mixture comprises anucleotide cognate of a first base type and a nucleotide cognate of asecond base type, and wherein the second mixture comprises a nucleotidecognate of the first base type and a nucleotide cognate of a third basetype.

Embodiment 73

The method of embodiment 72, wherein a third mixture is contacted withthe primed template nucleic acid, the third mixture comprising anucleotide cognate of the second base type and a nucleotide cognate of afourth base type.

Embodiment 74

The method of embodiment 72, wherein a fourth mixture is contacted withthe primed template nucleic acid, the fourth mixture comprising anucleotide cognate of the third base type and a nucleotide cognate ofthe fourth base type.

Embodiment 75

The method of embodiment 74, wherein the first mixture lacks nucleotidecognates of the third and fourth base types, wherein the second mixturelacks nucleotide cognates of the second and fourth base types, whereinthe third mixture lacks nucleotide cognates of the first and third basetypes, and wherein the fourth mixture lacks nucleotide cognates of thefirst and second base types.

Embodiment 76

The method of embodiment 71, wherein the first mixture comprises alabeled nucleotide cognate of a first base type and a labeled nucleotidecognate of a second base type, and wherein the first mixture comprises anon-labeled nucleotide cognate of a third or fourth base types.

Embodiment 77

The method of embodiment 76, wherein the second mixture comprises alabeled nucleotide cognate of the first base type and a labelednucleotide cognate of the third base type, wherein the second mixturecomprises a non-labeled nucleotide cognate of the second or fourth basetypes.

Embodiment 78

The method of embodiment 77, wherein a third mixture is contacted withthe primed template nucleic acid, the third mixture comprising a labelednucleotide cognate of the second base type and a labeled nucleotidecognate of the fourth base type, wherein the third mixture comprises anon-labeled nucleotide cognate of the first or third base types.

Embodiment 79

The method of embodiment 78, wherein a fourth mixture is contacted withthe primed template nucleic acid, the fourth mixture comprising alabeled nucleotide cognate of the third base type and a labelednucleotide cognate of the fourth base type, wherein the fourth mixturecomprises a non-labeled nucleotide cognate of the first or second basetypes.

Embodiment 80

The method of embodiment 71, wherein step (a) comprises sequentiallycontacting the primed template nucleic acid with at least four mixturesunder ternary complex stabilizing conditions, wherein each of themixtures comprises a polymerase and nucleotide cognates for at least twoof four different base types in the primed template nucleic acid,wherein the mixtures differ by at least one type of nucleotide cognate.

Embodiment 81

The method of embodiment 80, wherein the next correct nucleotide isidentified as a cognate of one of the four different base types ifternary complex is detected in at least two of the mixtures.

Embodiment 82

The method of embodiment 81, wherein each of the mixtures comprisesnucleotide cognates for at least two and no more than three of the fourdifferent base types in the primed template nucleic acid.

Embodiment 83

The method of embodiment 81, wherein each of the mixtures comprisesnucleotide cognates for at least two and no more than two of the fourdifferent base types in the primed template nucleic acid.

Embodiment 84

The method of embodiment 81, wherein each of the mixtures comprisesnucleotide cognates for at least three and no more than three of thefour different base types in the primed template nucleic acid.

EXAMPLES

The following examples describe several different configurations thatutilize disambiguation and/or imputation to identify nucleotides atindividual positions of nucleic acids.

A primed template nucleic acid is attached to a solid support in a flowcell. Reagents are delivered to the flow cell under conditions forstabilizing formation of a ternary complex between the primed template,polymerase and next correct nucleotide. The tables below refer to areagent delivery as a ‘flow.’ The number of reagent flows andcomposition of each reagent flow can vary as specified for eachconfiguration below. Furthermore, the reagents listed for each flow canbe delivered simultaneously or sequentially.

A stabilized ternary complex that forms on the solid support can includea fluorescent label on either the polymerase or nucleotide, as specifiedin the individual configurations below. Examinations are carried out todetect fluorescent signals on the solid support. The flow cell canoptionally be washed between each flow and examination to removebackground label and allow better signal to noise in detectingstabilized ternary complex formed on the solid support. Ternarycomplexes are stabilized and examined using techniques and apparatus setforth in U.S. Pat. App. Pub. No. 2017/0022553 A1 or U.S. Pat. App. Ser.Nos. 62/447,319; 62/440,624 or 62/450,397, each of which is incorporatedherein by reference.

Exemplary advantages for each configuration are set forth below. It willbe understood that reducing the number of detection channels generallyallows use of more affordable detection apparatus, faster imageacquisition time and in some cases higher resolution. Reducing thenumber of flows can provide for faster overall cycle time (i.e. thecumulative fluidic and detection time to interrogate each position inthe current example), lower overall cost of reagents and reduced volumeof fluidic waste. Reducing the number of different nucleotides canprovide lower cost for completing a cycle, reduced overall volume ofreagents during shipment and storage, and reduced volume of fluidicwaste.

One Color, Three Nucleotide Types, Three Deliveries

As shown in the first column of Table 1, three flows can be carried out,each to deliver a polymerase and one nucleotide type to the primedtemplate. In each case either the polymerase or the nucleotide can beattached to a fluorescent label. Examinations are carried out after eachflow. The fluorescent label can be the same for all flows. The signalexpected for a stabilized ternary complex formed with respectivenucleotide types, A, G, C and T, are indicated in the last four columns.A positive sign indicates that a fluorescent signal is detected and anegative sign indicates absence of significant signal. As is evidentfrom Table 1, the presence of a ternary complex where the next correctnucleotide is A, G or C can be determined from a signal detectedfollowing the flow where the respective nucleotide was delivered. Thenucleotide that was not delivered (i.e. the T nucleotide in thisexample) is imputed from the absence of significant signal detected inall three examination steps. Note that absence of signal for T or anyother nucleotide may be due to absence of the nucleotide in the flow.Alternatively, the non-detected nucleotide may be present in the flowand capable of forming ternary complexes, albeit ternary complexes thatare not detectable (e.g. due to absence of a label on the ternarycomplex formed with that nucleotide).

TABLE 1 Step A G C T Flow pol + A (+) (−) (−) (−) 1^(st) Exam Flow pol +G (−) (+) (−) (−) 2^(nd) Exam Flow pol + C (−) (−) (+) (−) 3^(rd) Exam

An advantage of the configuration in Table 1 is that four nucleotidescan be distinguished using only one label, a single detection channel(i.e. excitation and emission collection at the same wavelength for theproducts of all flows), only three reagent delivery steps, only threeexamination steps and only three nucleotide types.

One Color, Three Nucleotide Types, Two Deliveries

As shown in the first column of Table 2, two flows can be carried out,to deliver a total of three nucleotide types to the primed template.Either the polymerase or the nucleotide can be attached to a fluorescentlabel. Examinations are carried out after each flow. The fluorescentlabel can be the same for both flows. The signal expected for astabilized ternary complex formed in the first flow (and detected in the1^(st) exam) will indicate that a ternary complex has been formed butwill be ambiguous regarding whether the complex contains an A or G asthe next correct nucleotide. The signal expected for a stabilizedternary complex formed in the second flow (and detected in the 2^(nd)exam) will indicate that a ternary complex has been formed but will beambiguous regarding whether the complex contains an A or C as the nextcorrect nucleotide. As is evident from comparison of signals in Table 2for the two examinations, the presence of a ternary complex where thenext correct nucleotide is A, G or C can be determined by disambiguationwhereby A is indicated by signal in both examinations, G is indicated bysignal in the 1^(st) exam and absence of significant signal in the2^(nd) examination, and C is indicated by absence of significant signalin the 1^(st) examination and detection of signal in the 2^(nd) exam.The nucleotide that was not delivered (i.e. the T nucleotide in thisexample) is imputed from the absence of significant signal in both ofthe examinations. Note that absence of signal for T or any othernucleotide may be due to absence of the nucleotide in the flow.Alternatively, the non-detected nucleotide may be present in the flowand capable of forming ternary complexes, albeit ternary complexes thatare not detectable (e.g. due to absence of a label on the ternarycomplex formed with that nucleotide).

TABLE 2 Step A G C T Flow pol + A + G (+) (+) (−) (−) 1^(st) Exam Flowpol + A + C (+) (−) (+) (−) 2^(nd) Exam

An advantage of the configuration in Table 2 is that four nucleotidescan be distinguished using only one label, a single detection channel,only two reagent delivery steps, only two examination steps, and onlythree different nucleotides.

Two Colors, Six Nucleotide Types, Two Deliveries

Table 3 shows a configuration in which two flows are carried out, todeliver nucleotide types having four different bases. However, theternary complexes that form with two of the bases have alternativelabels in either flow. Specifically, ternary complexes that form withthe G nucleotide will be red in the first flow and blue in the secondflow. Ternary complexes that form with the T nucleotide will be blue inthe first flow and red in the second flow. As such, this configurationis carried out using six different nucleotide types. The nucleotides canbe attached to the different fluorescent labels in the mixturesexemplified in the first column of Table 3. Examinations are carried outafter each flow. The signal expected for a stabilized ternary complexformed in the first flow (and detected in the 1^(st) exam) will indicatethat a ternary complex has been formed, but a signal detected in the redchannel will be ambiguous regarding whether the complex contains an A orG as the next correct nucleotide and a signal detected in the bluechannel will be ambiguous regarding whether the complex contains a C orT as the next correct nucleotide. The signal expected for a stabilizedternary complex formed in the second flow (and detected in the 2^(nd)exam) will indicate that a ternary complex has been formed but a signaldetected in the red channel will be ambiguous regarding whether thecomplex contains an A or T as the next correct nucleotide and a signaldetected in the blue channel will be ambiguous regarding whether thecomplex contains a G or C nucleotide. As is evident from comparison ofsignals in Table 3 for the two examinations, the next correct nucleotidecan be identified by disambiguation whereby A is indicated by a redsignal in both examinations, G is indicated by a red signal in the1^(st) exam and a blue signal in the 2^(nd) examination, C is indicatedby a blue signal in the 1^(st) examination and a blue signal in the2^(nd) exam, and T is indicated by a blue signal in the 1^(st)examination and a red signal in the 2^(nd) examination.

TABLE 3 Step A G C T Flow pol + A_(red) + G_(red) C_(blue) + T_(blue)red red blue blue 1^(st) Exam Flow pol + A_(red) + G_(blue) + C_(blue) +T_(red) red blue blue red 2^(nd) Exam

An advantage of the configuration in Table 3 is that four nucleotidescan be distinguished using only two labels, only two detection channels,only two reagent delivery steps, and only two examination steps.Although six different nucleotides are used in this configuration, anadded benefit is improved error checking for all types of nucleotides inthe template due to the fact that two different positive signals aredetected for each type of next correct nucleotide at a particularposition in the template.

The configuration in Table 3 can be modified to use intensity scaling,instead of wavelength differences, to distinguish stabilized ternarycomplexes. For example, the red labels can be retained and the bluelabels can be replaced with red labels that have a fraction of theintensity of the red labels that are retained. An advantage of thismodification is that two channel detection can be replaced with simplerand cheaper single channel detection (so long as signal intensities canbe distinguished in the single channel).

Two Colors, Four Nucleotide Types, Two Deliveries

Table 4 shows a configuration in which two flows are carried out, todeliver a total of four nucleotide types to the primed template. Thenucleotides can be attached to the different fluorescent labels in themixtures exemplified in the first column of Table 4. Examinations arecarried out after each flow. The signal expected for a stabilizedternary complex formed in the first flow (and detected in the 1^(st)exam) will indicate that a ternary complex has been formed, but a signaldetected in the red channel will be ambiguous regarding whether thecomplex contains an A or G as the next correct nucleotide and a signaldetected in the blue channel will be ambiguous regarding whether thecomplex contains a C or T as the next correct nucleotide. A red signaldetected in the 2^(nd) exam will indicate that A is the next correctnucleotide in the ternary complex and a blue signal will indicate that Cis the next correct nucleotide in the ternary complex. As is evidentfrom comparison of signals in Table 4 for the two examinations, the nextcorrect nucleotide can be identified by disambiguation whereby A isindicated by a red signal in both examinations, G is indicated by a redsignal in the 1^(st) exam and absence of significant signal in the2^(nd) examination, C is indicated by a blue signal in bothexaminations, and T is indicated by a blue signal in the 1^(st)examination and absence of significant signal in the 2^(nd) examination.

TABLE 4 Step A G C T Flow pol + A_(red) + G_(red) + C_(blue) + T_(blue)red red blue blue 1^(st) Exam Flow pol + A_(red) + C_(blue) red (−) blue(−) 2^(nd) Exam

An advantage of the configuration in Table 4 is that four nucleotidescan be distinguished using only two labels, only two detection channels,only two reagent delivery steps, and only two examination steps. Fournucleotide types are used in this configuration, but error checking isprovided for two of the nucleotide types in the template due to the factthat two different positive signals are detected for two next correctnucleotide types at a particular position in the template.

The configuration in Table 4 can be modified to use intensity scaling,instead of wavelength differences, to distinguish stabilized ternarycomplexes. For example, the red labels can be retained and the bluelabels can be replaced with red labels that have a fraction of theintensity of the red labels that are retained. An advantage of thismodification is that two channel detection can be replaced with simplerand cheaper single channel detection (so long as signal intensities canbe distinguished in the single channel).

Two Colors, Three Nucleotide Types, Two Deliveries

Table 5 shows a configuration in which two flows are carried out, todeliver a total of three nucleotide types to the primed template. Thenucleotides can be attached to the different fluorescent labels in themixtures exemplified in the first column of Table 5. Examinations arecarried out after each flow. The signal expected for a stabilizedternary complex formed in the first flow (and detected in the 1^(st)exam) will indicate that a ternary complex has been formed, but a signaldetected in the red channel will be ambiguous regarding whether thecomplex contains an A or G as the next correct nucleotide. A blue signalin the 1^(st) examination will indicate that C is the next correctnucleotide. A red signal detected in the 2^(nd) exam will indicate thatA is the next correct nucleotide in the ternary complex and a bluesignal will indicate that C is the next correct nucleotide in theternary complex. As is evident from comparison of signals in Table 4 forthe two examinations, the next correct nucleotide can be identified bydisambiguation whereby A is indicated by a red signal in bothexaminations, G is indicated by a red signal in the 1^(st) exam andabsence of significant signal in the 2^(nd) examination, and C isindicated by a blue signal in both examinations. The nucleotide that wasnot delivered (i.e. the T nucleotide in this example) is imputed fromthe absence of significant signal in both of the examinations. Note thatabsence of signal for T or any other nucleotide may be due to absence ofthe nucleotide in the flow. Alternatively, the non-detected nucleotidemay be present in the flow and capable of forming ternary complexes,albeit ternary complexes that are not detectable (e.g. due to absence ofa label on the ternary complex formed with that nucleotide).

TABLE 5 Step A G C T Flow pol + A_(red) + G_(red) + C_(blue) red redblue (−) 1^(st) Exam Flow pol + A_(red) + C_(blue) red (−) blue (−)2^(nd) Exam

An advantage of the configuration in Table 5 is that four nucleotidescan be distinguished using only two labels, only two detection channels,only two reagent delivery steps, only two examination steps and onlythree nucleotide types. Error checking is provided for two of thenucleotide types in the template due to the fact that two differentpositive signals are detected for two next correct nucleotide types at aparticular position in the template.

The configuration in Table 5 can be modified to use intensity scaling,instead of wavelength differences, to distinguish stabilized ternarycomplexes. For example, the red labels can be retained and the bluelabels can be replaced with red labels that have a fraction of theintensity of the red labels that are retained. An advantage of thismodification is that two channel detection can be replaced with simplerand cheaper single channel detection (so long as signal intensities canbe distinguished in the single channel).

Three Color Detection Schemes

Tables 6 through 8 show several configurations that exploit threedifferent labels detected in three different channels. The configurationin Table 6 uses only three nucleotide types, only three labels andimputation of one unused nucleotide type, thereby providing advantagesof requiring no more than one flow and fewer labels (and detectionchannels) than the number of nucleotides distinguished. Note thatabsence of signal for T may be due to absence of the T nucleotide in theflow. Alternatively, the T nucleotide may be present in the flow andcapable of forming ternary complexes that are not detectable (e.g. dueto absence of a label on the ternary complex formed with the Tnucleotide).

TABLE 6 Step A G C T Flow pol + A_(red) + G_(yellow) + C_(blue) redyellow blue (−) 1^(st) Exam

The configuration in Table 7 uses four nucleotide types, two flows andonly three labels, thereby providing an advantage of requiring fewerlabels (and detection channels) than the number of nucleotidesdistinguished. As a further advantage, error checking is provided forthree of the nucleotide types in the template due to the fact that threedifferent positive signals are detected at any position in the template.

TABLE 7 Step A G C T Flow pol + A_(red) + G_(yellow) + C_(blue) +T_(red) red yellow blue red 1^(st) Exam Flow pol + A_(red) +G_(yellow) + C_(blue) red yellow blue (−) 2^(nd) Exam

Table 8 shows a configuration that uses two flows and only three labels,thereby providing an advantage of requiring fewer labels (and detectionchannels) than nucleotides distinguished. Although five nucleotide typesare used, error checking is provided for all four nucleotide types inthe template due to the fact that four different positive signals aredetectable at any position in the template.

TABLE 8 Step A G C T Flow pol + A_(red) + G_(yellow) + C_(blue) +T_(red) red yellow blue red 1^(st) Exam Flow pol + A_(red) +G_(yellow) + C_(blue) + T_(blue) red yellow blue blue 2^(nd) Exam

The configurations in Table 6 through 8 can be modified to use intensityscaling, instead of wavelength differences, to distinguish stabilizedternary complexes. For example, the red labels can be retained and theblue and yellow labels can be replaced with red labels that have onethird and 2 thirds, respectively, of the intensity of the retained redlabels. An advantage of this modification is that three channeldetection can be replaced with simpler and cheaper single channeldetection (so long as signal intensities can be distinguished in thesingle channel).

Repetitive Examination of Cognate Nucleotides

The flows and exams shown in Tables 1 through 8 can be repeated prior toperforming an extension step. The repetitions can lead to the flows andextensions being carried out at least 2, 3, 4, 5 or more times percycle. Accordingly, a particular position in a template can berepeatedly sampled for ability to form ternary complex with a particularnucleotide type. This repetition can yield a more accurate nucleotideidentification than may result absent the repetition. The repetition canalso provide a basis for statistical analysis of results and reportingof statistical variance or statistical confidence in the nucleotidecalls made at individual positions in a template nucleic acid.

As shown in Table 9, four flows can be carried out, each delivering adifferent combination of two different nucleotide types, and the resultof this combinatorial approach is to evaluate each of the fournucleotide types twice. Either the polymerase or the nucleotide can beattached to a fluorescent label. Examinations are carried out after eachflow. The fluorescent label can be the same for both flows. The signalexpected for a stabilized ternary complex formed in the first flow (anddetected in the 1^(st) exam) will indicate that a ternary complex hasbeen formed but will be ambiguous regarding whether the complex containsan A or G as the next correct nucleotide. The signal expected for astabilized ternary complex detected in the 2^(nd) exam will be ambiguousregarding whether the complex contains an A or C as the next correctnucleotide. The signal expected for a stabilized ternary complexdetected in the 3^(rd) exam will be ambiguous regarding whether thecomplex contains a G or T as the next correct nucleotide. The signalexpected for a stabilized ternary complex detected in the 4^(th) examwill be ambiguous regarding whether the complex contains a C or T as thenext correct nucleotide.

TABLE 9 Step A G C T Flow pol + A + G (+) (+) (−) (−) 1^(st) Exam Flowpol + A + C (+) (−) (+) (−) 2^(nd) Exam Flow pol + G + T (−) (+) (−) (+)3^(rd) Exam Flow pol + C + T (−) (−) (+) (+) 4^(th) Exam

As is evident from comparison of signals in Table 9 for the fourexaminations, the presence of a ternary complex where the next correctnucleotide is A, G, C or T can be determined by disambiguation whereby Ais indicated by signal in 1^(st) and 2^(nd) examinations, G is indicatedby signal in the 1^(st) and 3^(rd) examinations, C is indicated bysignal in 2^(nd) and 4^(th) examinations, and T is indicated by signalin 3^(rd) and 4^(th) examinations.

An advantage of this configuration is that each nucleotide type isobserved two times per template position (i.e. two times per sequencingcycle). This in turn improves accuracy compared to a configuration whereonly a single observation is made for each nucleotide type per cycle.Flowing two nucleotides at a time improves speed and reduces reagentcost compared to a configuration where 8 flows and 8 exams are carriedout to achieve discrete detection of the 8 individual ternary complexesper cycle.

As shown in Table 10, four flows can be carried out, each delivering amixture of three nucleotide types, such that all four nucleotide typesare evaluated three times each. Again, the polymerase or the nucleotidecan be attached to a fluorescent label and examinations are carried outafter each flow. The fluorescent label can be the same for both flows.The signal expected for a stabilized ternary complex formed in the firstflow (and detected in the 1^(st) exam) will indicate that a ternarycomplex has been formed but will be ambiguous regarding whether thecomplex contains an A, G or C as the next correct nucleotide. The signaldetected in the 2^(nd) exam will be ambiguous regarding whether thecomplex contains a G, C or T as the next correct nucleotide. The signaldetected in the 3^(rd) exam will be ambiguous regarding whether thecomplex contains an A, C or T as the next correct nucleotide. The signaldetected in the 4^(th) exam will be ambiguous regarding whether thecomplex contains an A, G or T as the next correct nucleotide.

TABLE 10 Step A G C T Flow pol + A + G + C (+) (+) (+) (−) 1^(st) ExamFlow pol + G + C + T (−) (+) (+) (+) 2^(nd) Exam Flow pol + A + C + T(+) (−) (+) (+) 3^(rd) Exam Flow pol + A + G + T (+) (+) (−) (+) 4^(th)Exam

As is evident from comparison of signals in Table 10 for the fourexaminations, the presence of a ternary complex where the next correctnucleotide is A, G, C or T can be determined by disambiguation whereby Ais indicated by signal in 1^(st), 3^(rd) and 4th examinations, G isindicated by signal in the 1^(st), 2^(nd) and 4^(th) examinations, C isindicated by signal in 1^(st), 2^(nd) and 3^(rd) examinations, and T isindicated by signal in 2^(nd), 3^(rd) and 4^(th) examinations.

An advantage of this configuration is that each nucleotide type isobserved three times per template position (i.e. two times persequencing cycle). This in turn improves accuracy compared to aconfiguration where only a single or double observation is made for eachnucleotide type per cycle. Flowing three nucleotides at a time improvesspeed and reduces reagent cost compared to a configuration where 12flows and 12 exams are carried out to achieve discrete detection of the12 individual ternary complexes per cycle.

In a variation on the examples shown in Tables 9 and 10, the ternarycomplexes that are detected can be distinguishable in each exam based onthe type of nucleotide that is present in the complex. Taking as anexample Table 9, the A and T nucleotides can form ternary complexeshaving a red label, whereas the G and C nucleotide can form ternarycomplexes having blue labels. Using two labels in this way will allowthe two ternary complexes that result from each flow to be distinguishedfrom each other in each exam. Similarly, intensity scaling can be usedto distinguish different types of ternary complexes in each exam. Assuch, disambiguation is not necessary and instead one type of ternarycomplex can be distinguished from the other in each exam to improveaccuracy and ease of data analysis.

Note that absence of signal for the non-detected nucleotides during eachexam (e.g. C and T in the 1^(st) exam of Table 9) may be due to absenceof those nucleotides in the flow. Alternatively, the non-detectednucleotides may be present in the flow and capable of forming ternarycomplexes that are not detectable (e.g. due to absence of a label on theternary complex formed with those nucleotides).

Throughout this application various publications, patents and/or patentapplications have been referenced. The disclosures of these documents intheir entireties are hereby incorporated by reference in thisapplication.

A number of embodiments have been described. Nevertheless, it will beunderstood that various modifications may be made. Accordingly, otherembodiments are within the scope of the following claims.

What is claimed is:
 1. A method of nucleic acid detection, comprising steps of: (a) contacting a primed template nucleic acid with a polymerase and a first mixture of nucleotides under conditions for forming a ternary complex at a nucleotide position in the template, wherein the first mixture comprises a nucleotide cognate of a first base type comprising a label that produces a first signal; a nucleotide cognate of a second base type comprising a label that produces the first signal; a nucleotide cognate of a third base type comprising a label that produces a second signal; and a nucleotide cognate of a fourth base type comprising a label that produces the second signal; (b) contacting the primed template nucleic acid with a polymerase and a second mixture of nucleotides under conditions for forming a ternary complex at the nucleotide position in the template, wherein the second mixture comprises a nucleotide cognate of the first base type comprising a label that produces the first signal; a nucleotide cognate of a second base type comprising a label that produces the second signal; a nucleotide cognate of a third base type comprising a label that produces the second signal; and a nucleotide cognate of the fourth base type comprising a label that produces the first signal; and (c) examining products of steps (a) and (b) for signals produced by a ternary complex that comprises the primed template nucleic acid, a polymerase and a next correct nucleotide.
 2. The method of claim 1, wherein the signals for the products of step (a) are acquired by a detector that is also used to detect the signals for the products of step (b).
 3. The method of claim 1, further comprising (d) adding a reversibly terminated, next correct nucleotide to the primer of the primed template nucleic acid after step (c), thereby producing an extended, reversibly terminated primer.
 4. The method of claim 3, further comprising repeating steps (a) through (c) for the primed template nucleic acid that comprises the extended, reversibly terminated primer.
 5. The method of claim 4, further comprising (e) removing the reversible terminator moiety from the extended, reversibly terminated primer after steps (a) through (c) are repeated.
 6. The method of claim 1, wherein the steps are carried out for a plurality of primed template nucleic acids having different sequences.
 7. The method of claim 6, wherein the plurality of primed template nucleic acids is attached to an array.
 8. The method of claim 1, wherein the examining of the products of step (a) is carried out prior to step (b).
 9. The method of claim 1, further comprising (i) contacting the primed template nucleic acid with a polymerase and a third mixture of nucleotides under conditions for forming a ternary complex at the nucleotide position in the template, wherein the third mixture comprises a nucleotide cognate of the second base type and a nucleotide cognate of a fourth base type; (ii) contacting the primed template nucleic acid with a polymerase and a fourth mixture of nucleotides under conditions for stabilizing a ternary complex at the nucleotide position in the template, wherein the fourth mixture comprises a nucleotide cognate of the third base type and a nucleotide cognate of the fourth base type; and (iii) examining products of steps (i) and (ii) for signals produced by a ternary complex that comprises the primed template nucleic acid, a polymerase and a next correct nucleotide.
 10. A method of nucleic acid detection, comprising: (a) sequentially contacting a primed template nucleic acid with first and second mixtures under conditions for forming a ternary complex, wherein each of the mixtures comprises a polymerase and nucleotide cognates for at least two of four different base types in the primed template nucleic acid, wherein the first mixture comprises a nucleotide cognate of a first base type comprising a label that produces a first signal; a nucleotide cognate of a second base type comprising a label that produces the first signal; a nucleotide cognate of a third base type comprising a label that produces a second signal; and a nucleotide cognate of a fourth base type comprising a label that produces the second signal; and the second mixture comprises a nucleotide cognate of the first base type comprising a label that produces the first signal; a nucleotide cognate of a second base type comprising a label that produces the second signal; a nucleotide cognate of a third base type comprising a label that produces the second signal; and a nucleotide cognate of the fourth base type comprising a label that produces the first signal; (b) examining the first and second mixtures, or products thereof, separately to detect ternary complexes; and (c) identifying the next correct nucleotide for the primed template nucleic acid molecule, wherein the next correct nucleotide is identified as a cognate of one of the four different base types if ternary complex is detected in the two mixtures.
 11. The method of claim 10, wherein step (a) comprises sequentially contacting the primed template nucleic acid with at least three mixtures under conditions for forming a ternary complex, wherein each of the mixtures comprises a polymerase and nucleotide cognates for four different base types in the primed template nucleic acid, wherein the mixtures differ by at least one type of nucleotide cognate.
 12. The method of claim 11, wherein the next correct nucleotide is identified as a cognate of one of the four different base types if ternary complex is detected in at least two of the mixtures.
 13. The method of claim 1, wherein the first and second signals comprise optical signals.
 14. The method of claim 13, wherein the first and second signals examined in step (c) are distinguished by different luminescence emission wavelengths.
 15. The method of claim 13, wherein the first and second signals examined in step (c) are distinguished by different luminescence excitation wavelengths.
 16. The method of claim 13, wherein the first and second signals examined in step (c) are distinguished by different luminescent intensities at a wavelength.
 17. The method of claim 5, further comprising repeating steps (a) through (e) to determine the nucleotide sequence for the template nucleic acid.
 18. The method of claim 10, further comprising (d) adding a reversibly terminated, next correct nucleotide to the primer of the primed template nucleic acid after step (b), thereby producing an extended, reversibly terminated primer.
 19. The method of claim 18, further comprising repeating steps (a) and (b) for the primed template nucleic acid that comprises the extended, reversibly terminated primer.
 20. The method of claim 19, further comprising (e) removing the reversible terminator moiety from the extended, reversibly terminated primer after steps (a) and (b) are repeated.
 21. The method of claim 18, wherein step (d) is carried out prior to step (c).
 22. The method of claim 10, wherein the steps are carried out for a plurality of primed template nucleic acids having different sequences.
 23. The method of claim 22, wherein the plurality of primed template nucleic acids is attached to an array. 